Isolation and Selection of Indigenous Azospirillum spp. and the IAA of Superior Strains Effects on Wheat Roots

World Journal of Agricultural Sciences 3 (4): 523-529, 2007
ISSN 1817-3047
© IDOSI Publications, 2007
Isolation and Selection of Indigenous Azospirillum spp.
and the IAA of Superior Strains Effects on Wheat Roots
1Gh. Abbas Akbari,


1Seyyed Mehdi Arab,


2H.A. Alikhani,

1I. Allahdadi and


3M.H. Arzanesh
Department of Agronomy and Plant Breeding, Aboureihan Paradise, University of Tehran, Iran
1
Department of Soil Science, Agricultural Paradise, University of Tehran, Iran
2
Soils and Water Research Center, Tehran, Iran
3
Abstract: IAA produced by bacteria of the genus Azospirillum spp. can promote plant growth by stimulating
root formation. Native Azospirillum spp., isolated from Irannian soils had been evaluated this ability in both
qualitative and quantitative methods and registered the effects of superior ones on morphological,
physiological and root growth of wheat. The roots of wheat seedling responded positively to the several
bacteria inoculations by an increase in root length, dry weight and by the lateral root hairs.
Key words: Azospirillum spp. % isolation % laboratory % qualitative % quantitative % wheat
INTRODUCTION
in the absence of tryptophan when grown aerobically
in the presence of NH4, while De Francesco et al. [15]
Some microorganisms of soil, like Azospirillum sp.
showed that the highest levels of auxin were produced in
Azotobacter sp; Enterobacter sp. etc. have shown to
both N2-fixing condition and limiting ammonia stationary
encourage plant growth, by promoting the outbreak of
cultures of A. brasilense strain Sp6. The pH has a
secondary roots. Bacteria of the genus Azopirillum
significant effect on the amount of IAA produced [16].
have been isolated from the rhizosphere and roots of a
Vitamins may also play a role in the regulation of IAA
variety of plants including cereals and grasses [1-4].
synthesis in A. brasilense. Very low levels of the B
Several reports have describbed the beneficial effect
vitamins, especially pyridoxine and nicotinic acid,
of Azospirillum inoculation on plant growth; hence
increased production of IAA in A. brasilense [17].
these organisms have been attracting interest [2, 3, 5].
It is possible to mimic the effects of Azospirillum
Inoculation with indigenous Azosprillum strains is an
using IAA which increases root hairs and branching [16].
important procedure when studying their inherent
Kolb and Martin [18] have shown that spraying a
capacity to benefit crops. In some cases, indigenous
solution of 10G9 g
lG1 IAA on roots of wheat growing in
strains can perform better than introduced strains in
root boxes resulted in a significant increase in root
promoting the growth of crops due to their superiour
length which mimicked Azospirillum inoculation.
adaptability to the environment. Inoculation with A.
Spraying an inoculum of A. brasilense strain FT-326
brasilense and a local strain clearly improved growth
on roots of Beta vulgaris resulted in significant
and increased the yield of three cultivars of wheat in
increases in both root length and number of laterals.
different areas of Israel [6].
Similarly, a cell-free supernatant of A. brasilense Cd

Azospirillum grown in culture are known to
applied to soybean plants induced the highest number
produce growth promoting compounds, such as
of roots and increased root length [19]. The work of
gibberllin-like and cytokinin-like substances and auxins
Jain and Partriquin [20] strongly suggests that IAA is
such as IAA from tryptophan [4, 7, 8 ]. The general
responsible for root hair branching in wheat, since it is
belief exists that Azospirillum increases root mass
affected by both plant and bacterial genomes. These
and function and changes root and root hair
genome effects can be explained by differential plant
morphology [9-13]. Horemans and Vlassak [14]
sensitivity to IAA and differing abilities of bacteria to
demonstrated that A. brasilense could produce IAA
produce IAA.
Corresponding Author: Dr. Gh. Abbas Akbari, Department of Agronomy and Plant Breeding, Aboureihan Paradise,
University of Tehran, Iran
523

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World J. Agric. Sci., 3 (4): 523-529, 2007
The possibility that roots produce plant growth-
into semisolid NFb medium (10 ml medium in a 20 ml-vial).
promoting substances in response to bacterial cell
After 72 h incubation at 33°C, the white halo formed
surface components or pectic enzymes of Azospirillum
3-6 mm below the media surface was a sign of
needs to be investigated but it is known that the effect
nitrogenase activity. When the cultures exhibited a
of Azospirillum on the formation of root hairs and
positive nitrogenase activity, they streaked out on
lateral roots is due not only to IAA but, probably, to still
CRA plates. Typical pink, often wrinkled colonies were
unidentified phytohormones or substances.
picked out and transferred into semi-solid NFb medium.
The first objective of this study was to isolate
Pellicle formation in this medium indicated successful
indigenous Azospirillum spp. from several dry lands
isolation. Purified colonies were transferred to a nutrient
on Tehran, Golestan and Khouzestan regions, Iran and
agar slant for storage and use for further studies.
to screen their IAA production ability (with both
qualitative and quantitative methods). The second was to
Qualitative IAA production ability test: This experiment
determine the effectiveness of superior strains, grown in
was carried out by the method proposed Bric et al. [23]:
different cultures - in promoting wheat root growth and
dry matter, using two methods, in order to select several
Growth media: Luria-Bertani (LB) agar medium was used
promising strains for further experiments.
containing: (g lG D.W.) Bacto-Tryptone (Difco), 10
1
;
yeast extract, 5; NaCl, 5; agar, 20. The PH was
MATERIALS AND METHODS
adjusted to 7.5 with 1 N NaOH before autoclaving. LB,
amended with 5 mM L-tryptophan, (LBT) (1.2115 g lG LB)
1
.
Isolation of Indigenous Azospirillum spp.: The media
used in this study were those recommended by Baldani
Assay conditions: Plates (9-cm diameter) containing LBT
and Dobereiner [21] and Rodriguez Caceres [22].
medium were divided into a grid pattern by a fine-marker.
Grid plates were inoculated by 50 indigenous strains of
N-free semisolid malate medium (NFb medium):
Azospirillum spp. in two replications, using sterile tooth
(g lG D.W.) DL-malic acid, 5; K
1
HPO
picks. Each inoculated plate was overlaid with an 82 mm
2
, 0.5; MgSO
4
. 7H O,


4
2
0.2; KOH, 4; NaCl, 0.1; CaCl
diameter nitrocellulose membrane. Plates were overlaid
2, 0.02; agar, 1.75; trace
element solution, 2 ml; alcoholic solution of Bromothymol
immediately after inoculation and incubated until colonies
Blue (5%), 2 ml; Fe EDTA, 4 ml; vitamin solution, 1 ml;
reached 0.5 to 2 mm in diameter. After an appropriate
NaOH to adjust the PH to 6.8. The trace element solution
incubation period, the membrane was removed from
contained: 200 mg Na
the plate and treated with Van Urk Salkowski reagent
2MoO4.2H2O; 235 mg MnSO



.H
4
O
2
;
280 mg H BO
(2% 0.5M FeCl in 35% perchloric acid) [24]. Membranes
3
;
3
8 mg CuSO .5H O; 24 mg ZnSO .7H O;


4
2



4
2
3
200 ml D.W. The vitamin solution contained: 10 mg biotin;
were saturated in a petri dish by overlaying on a reagent-
20 mg pyridoxine; 100 ml D.W.
saturated filter paper (whatman no. 2). The reaction was
allowed to proceed until adequate color developed. All
Congo Red Agar (CRA) medium: (g lG D.W.) DL-malic
1
reagent incubations were conducted at room temperature
acid, 5; K
at 30°C. Bacteria producing IAA were identified by the
2HPO4, 0.5; MgSO


4. 7H

2O, 0.2; KOH, 4.5;
NaCl, 0.1; agar, 15-20; yeast extract, 0.5; FeCl .6H
formation of a characteristic pink to red halo within the
3
O
2
,
0.015; 15 ml Congo red solution (0.25 %); NaOH to
membrane immediately surrounding the colony. Halo color
adjust the PH to 7.0.
and diameter were recorded after 30 min and 2 h (Fig. 1).
Azospirillum spp. were isolated from roots of
wheat (Triticum aestivum.), maize (Zea mays.), barley
Standard assays: A dilution series with concentrations
(Hordeum vulgare.), rye (Secale cereale.) grass (poa sp.)
of IAA (0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1, 2, 4 and 8 nmol) per
and some weeds (Cynodon dactylon, Dactylis sp.,
10 micro liter aliquot was applied directly onto
Lolium sp., Digitaria sp.). Root samples were collected
nitrocellulose membrane and were assayed on whatman
from Tehran (Karaj, Qazvin and Varamin), Golestan and
no. 2 filter pads saturated with Salkowski reagent. The
Khouzestan regions, Iran. Fresh root samples were
halo colors were compared with those of bacteria.
washed in rapidly running tap water for 5 min to remove
the soil particles adhering to the root surface. The roots
Quantitative IAA production ability test: To determine the
were rinsed in sterile water, then cut into pieces (5-8
amounts of IAA produced by each isolate, a colorimetric
mm),which were macerated with forceps and introduced
technique was performed using the Van Urk Salkowski
524

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World J. Agric. Sci., 3 (4): 523-529, 2007
Experiments testing the influences of the bacteria on
wheat root growth: Seeds (variety Roshan) were surface
sterilized by Ethanol (96%) for 10 seconds, Sodium
hydrochloride (5%) for 10-12 min and were washed with
sterile distilled water at least 7 times, then incubated at
25°C for germination. Germinated grain was placed in an
Eppendorf plastic tube of which the tip had been excised.
The plastic tube was placed in a 30 ml test tube on top of
10 ml of Hoagland Solution.The test tubes were
supplemented with 100 micro liter of a 2 day old culture
of Azospirillum spp. strains with the same bacterial
solution (McFarland). In the second method; one third
of a 30 ml test tube was filled with dry sand and 10 ml
of Hoagland Solution was added into it. After
Fig. 1: Visualization of colonies of Azospirillum on
autoclaving the grains were thrown in the tubes and
nitrocellulose membrane after reaction of a
inoculated with Azospirillum spp.
membrane with the Salkowski reagent
In order to compare the ability of the bacteria to
produce IAA, a dilution series with concentration of IAA
reagent. Isolates were grown in no-agar LBT medium and
(0.01, 0.02, 0.04, 0.06, 0.08, 0.1 and 0.2 nmol) were added to
incubated at a 30°C temperature during 24 h in a rotary
the Hoagland Solution. All manipulations were performed
shaker (90 rpm). After that time they were centrifuged
under sterile conditions. After incubating at 28°C for 14
(5000 rpm, 25 min). The supernatant liquid was mixed with
days (14 h in the light, 10 h in the dark per day), number of
salkowski reagent (2:1) and the color was measured by
roots, root length and dry weight were determined.
spectroscopy at 530 nm after 30 min and 2 h.
Using LB medium, concentrations of IAA (0, 5, 10, 15,
RESULTS AND DISCUSSION
20, 25, 30, 40, 60, 80, 100, 150, 200 and 300 ppm) were
prepared, treated with Salkowski reagent as above and
Isolation of Indigenous Azospirillum spp.: The
the developed color was measured.
inoculation of crop plants with associative N2-fixing
bacteria of the genus Azospirillum was proposed in the
Assays to test the stimulatory effects of Azospirillum
mid-1970s as a new approach to provide fixed N and to
spp. on wheat root growth:
reduce fertilization requirements or to increase yield [25].
Organisms and growth: The ten superior Azospirillum
Also the plant-growth-promoting abilities of Azospirillum
spp. strains were grown at 30°C for 24 h in 100 ml Erlen
have aroused interest in its use as bacterial fertilizer [26].
mayors containing 4 different mediums: (g lG D.W.)
1
I)
The initial parameter for the quality determination of
K2HPO ,
4 0.78; KH PO , 0.61; MgSO . 7H O, 0.2; FeSO .
inoculum formulations of agricultural use is the isolation


2
4

4
2


4
7H O,
2
0.00625; EDTA(=Tiriplex III), 0.0093;
and identification of the guaranteed microorganisms.
Na MoO
2
.2H
4
O,
2
0.02; MnSO .H O, 0.01; DL-malic acid,
About fifty Azospirillum strains were isolated from


4
2
5; NaOH to adjust the PH to 7.0; KNO3, 2.0. II) KNO



3 was
different parts of Tehran, Golestan and Khouzestan
replaced by 0.625
1
g l
G NH Cl. III, IV) the first and the
regions. All 50 strains were isolated from plant roots.

4
second mediums were supplemented with D,L tryptophan
(100 mg lG ). Sterile solution of biotin (10
1





G g
5
lG ) wa
1
s
Qualitative and Quantitative analysis of IAA production
added to the cultures.
by Azospirillum spp.: One of the principal mechanisms of
promoting plant growth is related to the capability of
Seed test: Eight native and commercial varieties of
Azospirillum to produce plant-growth-promoting
spring and winter wheat (Roshan, Adle jaded, Adle
substances [27]. Sixteen strains of Azospirillum spp.
ghadim, Bezostaya, Sardari, Inia, Ataei, Naz) were
whose colonies were immobilized on a nitrocellulose
obtained from the Gene Bank of the Agronomy and Plant
membrane and then treated with salkowski reagent,
Breeding Center. The seeds were tested for germination
produced a pink to red halo within the membrane
speed, uniform growth and number of radicles and the
surrounding the colonies (Table 1) and other bacteria not
suitable one was selected for the experiment.
producing IAA effected no color change in the membrane
525

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World J. Agric. Sci., 3 (4): 523-529, 2007
Fig. 2: Samples of the attained solutions of indole-3-acetics acid comparing with standards
Table 1: The results of qualitative and quantitative methods of producing
increase in intensity for a period of 30 min. Concentrations
IAA by bacteria of the genus Azospirillum
not visible at 30 min, did not develop upon further
Qualitative method
Quantitative method (ppm)
incubation.
-----------------------------------
-----------------------------------
Twenty five strains were selected for the quantitative
Bacteria
Diameter of colony Color
30 min
120 min
asseys including IAA producers and those which did not
T 4
1.00
A
123.2
48.2
produce Indolic compounds in the previous method.
T 6
2.66
C
385.0
139.2
All 25 treated strains in a culture medium containing
T 8
-
-
221.0
86.5
DL-Tryptophan source, produced IAA, as detected by
T 10
1.00
B
404.0
157.0
the salkowski reagent under colorimetry, in the range
T 11
1.33
C
266.0
96.0
29 mg lG to 761 ppm. Figure 2. shows samples of th
1
e
T 12
-
-
288.0
112.4
attained solutions of this compound comparing with
T 17
-
-
273.2
249.0
standards.
T 20
1.50
C
520.0
202.0
Bromus-I
-
-
190.0
74.0
The effects of Azospirillum spp. on root growth:
Dactylis-I
1.66
B
235.0
92.0
Among the 25 strains, an IAA detection test was
72 L
-
-
75.5
29.4
perfomed, 10 were selected for producing different
118-I
1.66
C
761.0
297.0
concentrations of IAA. Tables 2 & 3 show the effect of
Agri-II
1.00
B
463.0
181.0
different Azospirillum spp. culture supernatants on
166
1.00
A
193.9
75.5
wheat root number, length and dry weight in the two
122
-
-
337.0
131.5
methods (first: Hoagland, second: sand, assays). Data
7
1.60
C
51.0
20.0
are means of three replicates. Means in each column
171-I
1.50
C
183.0
71.4
130-II
2.00
C
169.0
66.0
followed by the same letter are not significantly different
161-II
-
-
379.0
262.6
at the 5% level according to Duncan’s multiple range
Kh-44
-
-
498.0
194.0
tests. Our results indicate that 14 day old wheat plants
Kh-8
1.13
B
446.0
174.0
had formed much longer roots and more root hairs and
Kh-9-II
-
-
313.0
122.0
lateral roots after the inoculation with Azospirillum spp.
124
1.40
C
29.0
11.0
In all media, 10 super strains revealed different results.
171
1.50
C
293.0
114.0
The dry weight for roots excised afterwards gave
A: light pink, B: pink, C: light red
10.63 mg for 14 d and 3.47 mg for negative controls and
in the second method 39.87 and 20.00 mg, respectively
and were easily distinguishable by visual inspection
for the same time (Tables 2 & 3). All ten strains produced
(Fig. 1). Color development was first visible at the highest
concentratins that can stimulate the elongation of the
IAA concentration within minutes and continued to
root, parallel to the increase in IAA production, without
526

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World J. Agric. Sci., 3 (4): 523-529, 2007
Table 2: Effect of different culture supernatants of Azospirillum spp. isolates
Table 3:
Effect of different culture supernatants of Azospirillum spp.
on wheat root number, length and dry weight in Hoagland
isolates on wheat root number, length and dry weight in sand,
method. Data are means of three replicates. Means in each column
assys method. Data are means of three replicates. Means in each
followed by the same letter are not significantly different at the 5%
column followed by the same letter are not significantly different
level according to Duncan’s multiple range tests
at the 5% level according to Duncan’s multiple range tests
First Method
Second Method
-----------------------------------------------------------
----------------------------------------------------------
Root
Root
Root Dry
Root
Root
Root Dry
Bacteria
Media
No.
Length (mm)
Wt.(mg)
Bacteria
Media
No.
Length (mm)
Wt.(mg)
T6
I
5.00b-f
27.33r
19.17lm
T6
I
7.33b-g
29.33n-q
4.43mn
II
5.33a-f
50.67k
19.30lm
II
8.00a-e
29.00o-r
6.00g-i
III
4.67c-f
51.00jk
31.07d
III
8.33a-e
46.00c-e
4.83j-n
IV
4.33d-f
58.00d-f
26.17e-g
IV
6.67b-g
44.33d-f
4.43mn
Kh-8
I
3.33f
41.00o
31.00d
Kh-8
I
5.67d-g
32.33lm-o
2.73q
II
3.67ef
33.67p
22.13i-k
II
9.33a-c
30.33m-p
3.03q
III
4.33d-f
45.33mn
33.87bc
III
8.00a-e
38.33i-k
4.83j-n
IV
4.67c-f
55.00f-i
25.27f-h
IV
9.33abc
37.00jk
4.77k-n
Agri-II
I
6.00a-d
45.00mn
33.53b-d
Agri-II
I
5.33e-g
32.67l-n
2.73q
II
6.00a-d
46.33lm
34.37bc
II
6.33b-g
33.00lm
3.10pq
III
4.33d-f
50.33k
35.57b
III
6.33b-g
36.33jk
4.90j-m
IV
3.33f
59.00c-e
25.83e-g
IV
7.67b-f
39.67h-j
4.83j-n
Kh-9-II
I
5.00b-f
24.33s
27.53ef
Kh-9-II
I
6.33b-g
28.00p-t
5.67h-k
II
6.33a-d
44.00m-o
19.03lm
II
6.00c-g
25.33s-u
6.57gh
III
5.67a-e
46.33lm
27.03ef
III
4.33fg
42.67e-h
4.43mn
IV
5.00b-f
56.33e-h
24.10g-i
IV
8.00a-e
45.33c-e
5.73g-j
171
I
7.33a
24.00s
25.97e-g
171
I
5.33e-g
25.67r-u
4.57l-n
II
7.00ab
44.00m-o
14.93n
II
6.00c-g
26.67q-u
6.63 fg
III
6.00a-d
42.67no
25.70e-g
III
4.33fg
38.33i-k
5.50ij-l
IV
5.33a-f
51.33jk
23.07h-j
IV
4.00g
43.33d-g
4.77k-n
T10
I
5.67a-e
21.67st
18.60lm
T10
I
8.33a-e
30.67m-p
4.63l-n
II
6.00a-d
52.33i-k
22.50i-k
II
6.00c-g
28.67p-s
6.30g-i
III
6.33a-d
52.33i-k
28.30e
III
4.33 fg
45.67c-e
8.60bc
IV
5.33a-f
61.67bc
35.73b
IV
4.00g
46.00c-e
7.70de
124
I
4.67c-f
17.67u
17.60m
124
I
8.33a-e
21.33v
4.13m-o
II
6.00a-d
30.00qr
28.17e
II
7.00b-g
23.67uv
4.27m-o
III
5.00b-f
43.67m-o
26.10e-g
III
7.33b-g
35.67kl
5.97g-i
IV
5.67a-e
54.00h-j
31.97cd
IV
9.33a-c
40.67g-i
7.63de
118-I
I
6.67abc
31.67pq
20.47j-l
II
5.67a-e
62.33bcd
28.50e
118-I
I
6.67b-g
41.67f-i
7.43ef
III
5.67a-e
60.33bcd
30.93d
II
7.67b-f
39.00i-k
9.27b
IV
6.00abcd
75.33a
39.87a
III
6.00c-g
55.00b
10.60a
Dactylis-I
I
6.00abcd
20.33tu
17.70m
IV
8.67a-e
60.33a
10.63a
II
5.67a-e
49.33kl
26.73e-g
Dactylis-I
I
9.00a-d
31.00m-p
4.80k-n
III
6.67abc
54.67ghi
28.23e
II
8.67a-e
26.67q-u
6.23g-i
IV
6.00abcd
56.33efgh
38.27a
III
9.67ab
52.67b
7.80c-e
166
I
5.67a-e
19.67tu
14.77n
IV
11.33a
48.67c
8.40cd
II
7.00ab
46.33lm
22.60i-k
166
I
8.67a-e
30.00m-q
3.90n-p
III
6.00abcd
52.33ijk
27.43ef
II
7.33b-g
24.67tu
5.73g-j
IV
5.00b-f
57.67defg
34.10bc
III
9.00a-e
44.00d-g
6.33g-i
Control
6.00abcd
30.67q
20.00k-m
IV
9.33abc
46.33cd
7.43ef
I: Cells grown with KNO3., II: Cells grown with NH




4Cl.
Control
6.33b-g
17.33w
3.47opq
III: Cells grown with KNO3+L-TRP, IV: Cells grown with NH





4Cl+L-TRP
527

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World J. Agric. Sci., 3 (4): 523-529, 2007
inhibiting it. The addition of DL-Tryptophan to the
the influence of root exudates cannot be excluded from
medium significantly enhanced the root growth due to the
consideration. Auxin produced by Azospirillum may
higher IAA production by the cells. Excised roots of
particularly cause the formation of lateral roots as
wheat seedling respond positively to the addition of IAA
observed for several plants [30].
(different con.) by an increase in root length, dry weight
Many investigators Tchan and Kennedy [31] and
and by the formation of additional lateral roots. However,
Christiansen-Wengiger [32] have shown that bacterial-
high concentrations of IAA inhibited the growth of wheat
produced IAA caused the formation of nodule-like
segments (data not shown). Cells grown on both nitrogen
tumours at the root tips of non-legumes which is
sources increased the root dry weight significantly, but
probabely of interest to agriculture. The findings of this
NH +
4 seemed to be more efficient than nitrate. Both root
study provide significant evidence that IAA was
test methods were found to be appropriate for such
produced by Azospirillum spp. and that it causes clear
experiments. However, the second method was more
biological activity in root plants. The development of
suitable for detecting stimulatory effects of Azospirillum
techniques for the utilization of plant-growth-promoting
on the lateral roots. Furthermore, root outgrowth, i.e.
bacteria (including Azospirillum) in order to reduce rates
round, nodule-like tumours were observed on roots in
of fertilizer application should be recommended for
some treatments.
financial reasons and also to prevent environmental
Many PGPRs produce growth regulators such as
pollution by avoiding excessive applications of
auxins [28, 29]. In this study we analyzed one of the plant
industrially produced fertilizers to cultivated fields.
hormones, indole-acetic acid (IAA), in the bacterial
culture of the Azospirillum spp. with both qualitative and
REFERENCES
quantitative methods and observed that strain 118-I
produced the largest amount of IAA (285.51 mg lG )
1
1.
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