




































Highlights in BioScience
ISSN:2682-4043
DOI:10.36462/H.BioSci.202404

Research Article

Open Access

1 Department of Biotechnology Research, Micro-

biology Laboratory, Kyaukse, Myanmar.
2 Department of Zoology, Mandalay University,

Mandalay, Myanmar.

* To whom correspondence should be
addressed: khinthaemarrmm@gmail.com

Editor: Alsamman M. Alsamman, International
Center for Agricultural Research in the Dry Areas
(ICARDA), Cairo, Egypt.

Reviewer(s):
AbdulAziz Ascandari, Chemical and Biochemical
Sciences-Green Process Engineering, University
Mohammed VI Polytechnic, BenGuerir, Morocco.

Jean Legeay, Université de Lorraine, INRAE,
UMR IAM - Interactions Arbres-Microorganismes,
France.

Received: June 4, 2024

Accepted: October 11, 2024

Published: December 25, 2024

Citation: Marr KT, Wilbur ND, Yu SS, Ei
SL. Evaluation on multi-trait activities of
Azotobacter spp. from various water samples in
Mandalay environments. 2024 Dec. 25;7:bs202404

Copyright: © 2024 Marr et al.. This is an
open access article distributed under the terms
of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and supplementary materials.
Funding: The authors have no support or funding to
report.
Competing interests: The authors declare that they
have no competing interests.

Evaluation on multi-trait activities of Azotobacter spp. from various
water samples in Mandalay environments

Khin Thae Marr1
>< �, San San Yu1

>< �, Naw Dolly Wilbur2
>< �, Shun Lai Ei1 >< �

Abstract

Fourteen water samples were collected from different locations of Mandalay and
Kyaukse Township. Among them, eight bacterial isolates named A1 to A8 were
nominated as Azotobacter according to their colonial morphology, microscopic
morphology, and pigment production. According to the biochemical characteristics
and sequence analysis of isolated bacteria, they were Azotobacter chroococcum,
Azotobacter vinelandii, and Azotobacter beijerinckii. The phosphate solubilizing
of these isolates was observed from 2.3 to 2.6 SI, and A4 had the highest solubility
index. The potassium decomposing of these isolates was observed 2.5 to 3.8 SI, and A6
and A8 had the highest K-decomposing activity. The zinc solubilizing of these isolates
was observed at 2.3 to 3.5 SI, and A3 had the highest zinc solubilizing index. Screening
methods showed that all eight strains have nitrogen fixing activity and indole acetic
acid (IAA) producing activity. The antagonistic activity of all isolated strains was also
found against Pythium sp. and Fusarium oxysporum. The isolated Azotobacter sp. can
be used as biofertilizer in the agriculture sector, which can increase crop yields and
enhance soil fertility according to their plant growth promoting activities.

Keywords: Isolation, characterization, 16S rRNA gene, plant growth, Azotobacter, biofertilizer.

Introduction
Azotobacteraceae are heterotrophic gram-negative bacteria and a group of aerobic. The main

characteristic of Azotobacteraceae is the ability to fix nitrogen in the natural atmosphere, which is

vital in the nitrogen cycle. Azotobacter species are generally spherical on LB medium or oval on

glucose nitrogen free medium and motile (G-NFMM) [1]. Azotobacter can live as cysts and produce

enormous amounts of capsular slime. They can grow on any medium that has a suitable pH and an

organic carbon source [2]. Azotobacter produces blue white fluorescent, green, and yellow green

pigments [3]. The optimum temperature range of Azotobacter is between 20 and 30 oC, and the

optimum pH of Azotobacter is 6.5 in neutral to alkaline soil [4]. They can be found in the soil of

a number of crop plants and vegetative plants [5]. Azotobacter species is the preferred organism

because they are non-pathogenic and can stay as cysts in soil for many years [6]. Beijerinck studied

Azotobacter chroococcum isolated from the rhizosphere in Holland in 1901, and it was the first

species. Azotobacter agilis was also studied by Beijerinck [7]. Lipman found Azotobacter vinelandii

in 1903 and Azotobacter beijerinckii in 1904 from the rhizosphere [8]. Azotobacter vinelandii has the

ability to produce a yellow-green pigment that gives fluorescence under ultraviolet light. In alkaline

soils, marine sediments, and marsh water, Azotobacter can be generally found [9]. Biofertilizers,

also known as organic preparations or bioinoculants, contain microorganisms that promote nitrogen

fixation, phosphate solubilizing, and potassium decomposing for plant growth [10]. Azotobacter,

Azospirillum, and Rhizobium are beneficial microbes for crop production to use as biofertilizers

[11; 12]. Nowadays, many countries use biofertilizers that give advantages to economic activity. The

chemical fertilizers threaten environmental pollution and human health. The research was designed

to isolate Azotobacter sp. from various water samples, to characterize biochemical tests, identify the

isolates by 16S rRNA sequencing analysis, and study the multi-trait activities of isolated bacteria.

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mailto:khinthaemarrmm@gmail.com
https://orcid.org/0009-0005-9920-9385
mailto:sakurasan2007@gmail.com
https://orcid.org/0000-0003-2465-2866
mailto:2929852582@qq.com
https://orcid.org/0009-0004-4759-6638
mailto:shunlai.ei@gmail.com
https://orcid.org/0000-0003-2444-2439
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Marr et al., 2024 Evaluation on multi-trait activities of Azotobacter spp.

Materials and Methods
Study Area and Sample Collection

The bottles of water samples were collected from different
places in Mandalay Environs and Kyaukse Township, Myanmar
(Figure 1). The water samples were taken from 5 to 12 cm of
water depth. The plastic bottle (1 liter) was used to collect water
samples. Then, the water bottles were carried to the department
of biotechnology research to be studied. The samples were named
S1 to S14 (Table 1). The strains isolated from these samples were
named A1 to A8. Their sampling sites and different locations of
bacterial isolates were shown in Table 2.

Table 1. Water samples collected from different locations in Mandalay and
Kyaukse Township.

No. Samples Collection site Township

1 S1* Ayeyarwady river jetty3 Mandalay

2 S2 Kandawgyi lake site1 Mandalay

3 S3* Kandawgyi lake site2 Mandalay

4 S4* Kandawgyi lake site3 Mandalay

5 S5* Fish pond in Mahar Muni Image Mandalay

6 S6* Taw-dwin Lake Kyaukse

7 S7* Tone-long (southern fish lake) Kyaukse

8 S8* Tone-lone (northern fish-lake) Kyaukse

9 S9 SunYe Loch site1 Kyaukse

10 S10 SunYe Loch site 2 Kyaukse

11 S11 SunYe Loch site3 Kyaukse

12 S12* SunYe Loch site 4 Kyaukse

13 S13 Thamote Canal Kyaukse

14 S14 Zawgyi River Kyaukse

Isolation of Bacteria
To isolate bacteria, 5 ml of water was added to the sterile

test tube, and 5 ml of 0.9% NaCl solution was mixed with it.
The tubes with the water sample were kept standing for about
30 minutes, and 100 µl of water was spread on G-NFMM [13].
After inverting the GNFMM plates, they were incubated at 35 oC
for 48 hours. The bacterial colonies that appeared on G-NFMM
were purified by subculturing and incubated at 35 oC for 2 days.
The shapes and sizes of isolated bacteria were characterized by
colonial and microscopic morphology. Microscopic morphology
was checked by using gram staining method [14]. The single
isolated colony was stained and examined with a drop of oil
immersion under a microscope at 100 magnifications to check
purity.

Biochemical Characteristics of Isolated Bacteria
The isolated bacteria were characterized by using some bio-

chemical tests. Biochemical characteristics of selected bacteria
were studied according to Bergey's manual of determinative bac-
teriology [15]. These tests include the motility test, the catalase
test, the starch hydrolysis test, the citrate utilization test, the
methyl red test, the Voges-Proskauer test, the indole test, and the
triple sugar iron agar test.

Figure 1. Location map of study area, Mandalay environments, and Kyaukse

Township. (Source: Myanmar information management unit and google earth

pro)

Molecular Identification of Bacterial Isolates
The purified bacterial isolates were sent to a DNA sequenc-

ing service, Eurofins company in Germany, and the sequence
data of individual isolates were queried using BLAST in the na-
tional center for biotechnology information (NCBI) and GenBank
databases. Sequence alignment results showed the maximum
identities of each isolate and their relevant accession number.
Muscle was used to align sequences that were identified, and a
phylogenetic tree was constructed based on the neighbor join-
ing (NJ) and the number of differences method algorithms using
the MEGA 11 software. To acquire accession numbers, the nu-
cleotide sequences of isolated Azotobacter species were then sent
to GenBank. For further application, all isolates were maintained
at the microbiology laboratory at the department of biotechnology
research.

Screening on Nitrogen Fixing Activity by Indophenol Method
The bacterial strains were injected in the bottles that contain

G-NFMM broth and incubated for one week at 35oC. Then 1 ml
of sample was centrifuged at 10000 rpm for 20 minutes. Am-
monium concentration was measured by indophenol method by
taking the supernatant. Firstly, 0.5 ml of EDTA solution was
added to supernatant. Then 0.5 ml of p-buffer was added. After
adding 2.5 ml of nitroprusside reagent, 2.5 ml of hypochlorite
solution was added to the supernatant in a small bottle. The
reaction mixture was placed at room temperature for 3 hours and
ammonium was estimated by the color of the sample.

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Marr et al., 2024 Evaluation on multi-trait activities of Azotobacter spp.

Table 2. Bacteria isolates from different locations in Mandalay environments and Kyaukse Township.

No. Samples Strain Collection Site pH Temperature

1 S1 A1 96°03 21.745" E and 21°58 13.576" N 7.8 25°C

2 S3 A2 96°03 21.709"E and 21°57 07.664" N 7.3 25°C

3 S4 A3 96°03 21.470" E and 21°56 49.277"N 7.4 25°C

4 S5 A4 96°04 48.758" E and 21°57 09.017"N 7.6 25°C

5 S6 A5 96°07 44.652" E and 21°32 32.639" N 8.2 25°C

6 S7 A6 96°07 34.727" E and 21°33 40.007"N 8.4 25°C

7 S8 A7 96°07 34.588" E and 21°33" 40.333" N 8.3 25°C

8 S12 A8 96°13 28.758" E and 21°40 36.516" N 8.2 25°C

In this method, 0 mg/ml of ammonium concentration will
show yellow color, and 3 mg/ml ammonium concentration will
show green color.

Screening on Phosphate Solubilizing Activity
To determine the phosphate solubilizing activity of isolated

bacteria, national botanical research institute phosphate (NBRIP)
media was used. The single colony of bacteria was placed on
NBRIP media. After five days of incubation at 35oC, the colony
showed a zone of clearance around it. The halo-zone diameter
was recorded by the phosphate solubilizing index [16]. The
solubilizing index (SI) can be determined using the following
formula:

S I =
colony diameter + halozone

colony diameter

Screening on Potassium Decomposing Activity
To determine the decomposing of potassium, the eight bac-

terial strains were tested on K decomposing medium by plating
method. Pure colonies were spotted on K medium and incubated
at 35oC. After five days, the clear zone around the colony was
observed, and the diameters of the clear zone were recorded
[17]. The solubilizing index (SI) was calculated using the same
formula as the phosphate solubilizing activity.

Screening on Zinc Solubilizing Activity
To determine the solubilizing of zinc, zinc solubilizing medium

was used to test the eight bacterial strains. The single colony of
bacteria was dotted on medium at 35oC. After incubating them
for five days, clear zones around the colonies were recorded [18].
The SI was measured and calculated using the above formula.

Screening on IAA Producing Activity
The production of IAA by the isolated strains was determined

calorimetrically using Salkowski's reagent mixture [35% per-
chloric acid (50 ml); 0.5 M FeCl3 (1 ml)]. To test IAA quality,
isolated strains were aseptically cultured in G-NFMM plates
containing 0.5 mg L-tryptophan per ml, and then incubated at
35oC for 3 days. After incubating the plates, 2 ml of Salkowski's
reagent and one drop of orthophosphoric acid were added into
the plate medium at room temperature. The color turned pink,
which indicated IAA production [19; 20].

Table 3. Biochemical characterization of isolates

Biochemical Test A1 A2 A3 A4 A5 A6 A7 A8 C

Gram Staining - - - - - - - - -

Catalase + + + + + + + + -

Starch Hydrolysis - - - - - - - - -

Citrate Utilization + + + + + + + + -

Motility + + + + + + + + -

Methyl Red + + + - - + - - -

Voges-Proskauer + + + + + + + + -

Indole - + - - - - - - -

TSI agar

glucose + + + + + + + + -

lactose + + + + + + + + -

sucrose + + + + + + + + -

gas - - - - - - - - -

H2S - + - + + - + - -

Solubility Index

Phosphate 2.5 2.5 2.3 2.6 2.3 2.3 2.3 2.3 0

Potassium 2.5 3 3 3 3.5 3.8 3.5 3.8 0

Zinc 2.8 2.3 3.5 2.3 2.3 2.3 2.3 3 0

Screening on Antagonistic Activity
To determine the antagonistic activity, a total of eight bacte-

rial isolates were tested with Pythium sp. and Fusarium oxyspo-
rum on potato dextrose agar (PDA) medium by plating method.
Dual cultures (bacteria and fungus) were spotted separately on
opposite sides on potato dextrose agar medium and incubated at
25oC. After three weeks of incubation, an antagonistic effect was
observed. The zone diameter of inhibition on fungal growth was
determined and recorded.

Results
Study Area and Sample Collection

Collection sites and township of S1 to S14 samples are shown
in Table 1, and the results of strains A1 to A8 from 14 samples

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Marr et al., 2024 Evaluation on multi-trait activities of Azotobacter spp.

Figure 2. Colonial and Microscopic Morphology of Isolated Strains.

are shown in Table 2. The samples showed a pH range of 7-9
and a temperature of 25°C.

Isolation and Biochemical Characterization of Bacteria
The purified bacteria were observed for their colonial mor-

phology and microscopic morphology such as color, shape and
size (Figure 2). The isolated colonies were checked by gram
staining method under microscope that revealed and character-
ized as Azotobacter species. They were pale-yellow and mucoid
according to their morphology and colony sizes were 2 to 4 mm.
According to gram straining, they were gram negative, cocci and
their sizes were 2 to 4 µm. The isolated bacteria, A1 to A8 were
identified by biochemical characterization tests shown in Table
3.

Identification of Azotobacter spp. by 16S rRNA gene sequencing
Detection of 16S rRNA genes of eight isolated bacteria by gel

electrophoresis was shown in Figure S1. The isolated strains be-
longed to the genus Azotobacter according to molecular analysis.
The strain A1 showed 100% identity with Azotobacter chroococ-
cum in NCBI BLAST search while the strain A2 showed 99.77%
identity with the Azotobacter chroococcum, respectively. The
strain A5 revealed 100% similarity with Azotobacter vinelandii
strain IAM 15004 (NR 041039.1) (Figure 3). The 16S rRNA
gene sequences of A6 showed 99.52% similarity with Azotobac-
ter vinelandii, A7 showed 98.87% with Azotobacter beijerinckii,

Figure 3. Phylogentic tree of 16S rRNA gene sequences of the bacterial eight

isolated strain from water samples.

A8 showed 99.58% with Azotobacter chroococcum and A3 ex-
hibited 99.50% with Azotobacter chroococcum, respectively. The
accession numbers were MT658735 for A1, MT658739 for A3,
MT658736 for A5, MT658737 for A6 and MT658738 for A7, re-
spectively, after submitting to the GenBank. The closely related
sequences were aligned the number of differences method by
using MEGA 11 and the neighbor-joining tree was created [21].
The bootstrap (1000 replication) was used for a statistical support
for the nodes in phylogenetic tree constructed with the 16S rRNA
similarities (%). Figure 3 showed that the phylogenetic tree
analysis involved 26 nucleotide sequences was united through
Muscle using MEGA 11 software. Bootstrap values below 50%
are not indicated, fewer than 5% alignment gaps. A total of 757
positions in the final data were involved.

Screening on Nitrogen Fixing Activity by Indophenol method
When the activity of isolated strains that fix nitrogen was

screened by culturing in the G-NFMM broth, all isolates changed
the color (Figure 4). The potential of nitrogen fixation was
indicated by their color changing of all isolates.

Screening on Phosphate Solubilizing Activity
A1, A2, A4 gave higher halo zone diameter than A5 in NBRIP

media. Strain A3, A5, A6, A7, and A8 showed the same clear
zone in phosphate solubilizing activity test (Figure 5).

Screening on Potassium Decomposing Activity
In K-decomposing test, strain A6, A8 exhibited 3.8 mm clear

zone and the strains A5, A7 showed the same 3.5 mm clear zone.

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Marr et al., 2024 Evaluation on multi-trait activities of Azotobacter spp.

Figure 4. Screening of Azotobacter species nitrogen fixing activity.

Figure 5. Screening on phosphate solubilizing activity of Azotobacter.

Strains A2, A3, A4 showed 3 mm clear zone strain A1 showed
2.5 mm clear zone (Figure 6).

Screening on Zinc Solubilizing Activity
All strains showed clear zone around their colonies on zinc

agar plates after 5 days of incubation at temperature 35°C. Among
eight strains, A3 showed the maximum clear zone, and followed
by A8 and A1. Strains A2, A4, A5, A6, and A7 showed the same
clear zone (Figure 7).

Screening on IAA Producing Activity
IAA producing activities of all isolated strains were screened

by using GNFMM containing 0.5 mg/ml L-tryptophan and incu-
bated at 35oC for 3 days. After incubating the plates, Salkowski's
reagent was added into the culture plates. IAA producing activity
was indicated by the development of pink color (Figure 8).

Screening on Antagonistic Activity
The antifungal activity of isolated strains A1 to A8 showed

good antifungal activity against Pythrium sp. and did not show

Figure 6. Screening on zinc solubilizing activity of Azotobacter.

Figure 7. Screening on potassium decomposing activity of Azotobacter.

distinct activity against Fusarium sp. after 3 weeks incubation
(Figure S2).

Discussion
Azotobacter can be found in the rhizosphere of vegetables

and plantation crops [22]. All plants need nitrogen, phosphate,
potassium and zinc for its growth. However, plants can absorb
ammonia converted from nitrogen. Azotobacter is proficient to
convert nitrogen into ammonia, making it readily available for
plant absorption [23]. Azotobacter is beneficial nitrogen fixer.
Azotobacter bio-fertilizer benefits in agriculture to integrate ni-
trogen management. Rhizobium, Azotobacter, and Azospirillum
enrich nitrogen nutrition in N-deficient soils [24]. After the death
of the Azotobacter cell, cell proteins are mineralized in soil and
contribute to the nitrogen accessibility of crop plants [25]. Gib-
berellic acid and IAA produced by Azotobacter encourage plant
growth development. Antimicrobial compounds produced by
some species of Azotobacter are able to inhibit plant pathogens
and prevent economic losses [26]. In this research, eight iso-
lated Azotobacter strains were obtained from 14 water samples.
The bacteria were characterized by 16S rRNA gene sequencing
method to know exactly about the genus of Azotobacter sp. They
were A. chroococcum, A. beijerinckii,and A. vinelandii. Eight
strains were cultured in G-NFM medium containing BTB as in-
dicator and showed changing of the color from green to blue
that was an indication of nitrogen fixing activity. The isolated
strains A1 to A8 showed nitrogen-fixing activity on the nitrogen-
free medium. All eight strains showed phosphate solubilization
activity on NBRIP medium. The widest clear zone diameter
was found in A. chroococcum of all isolated strains in phosphate
solubilization. To convert insoluble phosphorus to an accessible
form, orthophosphate, the capacity of some microorganisms is an
important trait in a PGPR for increasing crop yields. Azotobacter
can also convert insoluble phosphate to orthophosphate that is
soluble for plants to absorb [27]. Insoluble inorganic phosphate
can be absorbed by the roots of plants with the help of bacteria
that can solubilize phosphate [28]. Eight isolates showed the
clear zone around the colonies in potassium decomposition.

A. vinelandii A6 and A. chroococcum A8 showed the highest
potassium solubilizing activity among eight strains. The potas-
sium is available in four forms i.e., K solution, K as mineral, K
as transferable metal and K as non-exchangeable metal. Min-
eral K held tightly on the surface of clay. Non-exchangeable

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Marr et al., 2024 Evaluation on multi-trait activities of Azotobacter spp.

Figure 8. Indole acetic acid (IAA) producing activity of Azotobacter sp.

K is a primary factor in determining soil and tightly bound in
the crystal lattice. Exchangeable K is loosely bound water in
the soil. Soluble K could be measured in a water extract of soil
[29; 30]. Zinc solubilizing bacteria showing clear halo zone
around their colonies were found in all eight strains. Azotobacter
chroococcum A3 showed the widest clear zone among the eight
strains. A very slight portion of total zinc is present as soluble
form in soil solution. The majority of zinc in soil is found in
insoluble compounds and minerals [31]. One of the important
hormones which promote plant growth is IAA. A positive cor-
relation was detected between tryptophan concentrations and
IAA production. All strains produced IAA in screening of IAA
production, using tryptophan as carrier. Fusarium oxysporum
and Pythium fungi were found to cause severe diseases in vari-
ous crops [32]. Pythium fungi are water molds, and their spores
attack living plants, causing issues such as seedling decay, root
and crown rot, and leaf blight [33; 34]. Fusarium oxysporum is a
fungal pathogen that infect banana, tomato, many other vegeta-
bles and ornamentals [35; 36]. In the present study, all strains
noticeably showed fungal growth inhibition against the tested
pathogen (Pythium). The antifungal activity of A. chroococcum
A1, A3 and A4, A. vinelandii A5 and A6, A. beijerinckii A7
were occurred against Pythium sp. A. chroococcum A2 and A8
were fair antifungal activity against Pythium sp. after 3 weeks.
A. chroococcum A4 and A8, the other strains were observed as
moderate antifungal activity. As for antifungal activity against
Fusarium oxysporum, all isolated strains exhibited minimal antag-
onistic activity, as indicated by the small zone areas. According
to the data, eight isolated strains showed good antifungal activity
against the Pythrium sp. although they have no good activity
against Fusarium sp. Azotobacter can be used as a bio-fertilizer
without any significant limitations or challenges, except that it
should not be used in conjunction with chemical pesticides and
herbicides. The optimum conditions such as pH and temperature
of isolated bacteria should be explored for their optimum growth
and plant growth promoting activities.

As for future research, isolated Azotobacter strains would
be used in combination with other bio-fertilizers and would be
used as antifungal and bio-fertilizers in agricultural fields. Bio-
fertilizers in Myanmar using Azotobacter have already been used
in agriculture [37]. Azotobacter can fix nearly 20 kg N/ha per

year to use as a substitute for fertilizer in crop production [38; 39].
Therefore, it can reduce the cost for mineral fertilizers. It has
economic impact and safety in farmers.

Conclusion
Eight bacterial isolates were obtained and identified as Azo-

tobacter vinelandii, Azotobacter beijerinckii and Azotobacter
chroococcum. Their existence of spherical form is about 2 µm
and that of oval form is about 3-4 µm. Their shapes were ranging
from cocci which occurred single, in pairs or irregular clusters
and some were arranged in chains of variable lengths. They were
motile, gram negative and cysts formed. All eight strains showed
nitrogen fixing, phosphate solubilizing, potassium decomposing,
zinc solubilizing and IAA producing activities. They also have
antifungal activity against Pythium and Fusarium oxysporum
fungi. Therefore, Azotobacter strains from this study could be
used to develop bio-fertilizer which can enhance soil fertility due
to their plant growth promoting activities.

Acknowledgement
We are greatly indebted to Dr. Thant Zin, Professor, Head of

the Department of Zoology, University of Mandalay, Ministry of
Education for allowing us to conduct the research. Our special
thanks to Dr. Aye Aye Khaing, Director General, Department of
Biotechnology Research, Ministry of Science and Technology
for allowing us to conduct the study in the laboratory. Thanks
to Dr. Tin Mar Lynn and all the researchers of Microbiology
Laboratory, Department of Biotechnology Research, Kyaukse for
their kind help and sharing their experiences.

Supplementary
Figure S1: Detection of 16S rRNA gene from isolated bacte-

ria by gel electrophoresis. Figure S2: Screening on antagonistic
activity of Azotobacter against A. Pythium sp. and B. Fusarium
sp.

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	Abstract
	Introduction
	Materials and Methods
	Study Area and Sample Collection
	Isolation of Bacteria
	Biochemical Characteristics of Isolated Bacteria
	Molecular Identification of Bacterial Isolates
	Screening on Nitrogen Fixing Activity by Indophenol Method
	Screening on Phosphate Solubilizing Activity
	Screening on Potassium Decomposing Activity
	Screening on Zinc Solubilizing Activity
	Screening on IAA Producing Activity
	Screening on Antagonistic Activity

	Results
	Study Area and Sample Collection
	Isolation and Biochemical Characterization of Bacteria
	Identification of Azotobacter spp. by 16S rRNA gene sequencing
	Screening on Nitrogen Fixing Activity by Indophenol method
	Screening on Phosphate Solubilizing Activity
	Screening on Potassium Decomposing Activity
	Screening on Zinc Solubilizing Activity
	Screening on IAA Producing Activity
	Screening on Antagonistic Activity

	Discussion
	Conclusion
	Supplementary

