Advances in Environmental Biology Azotobacter Chrococcum Fars Province

Advances in Environmental Biology, 8(17) September 2014, Pages: 837-841
AENSI Journals
Advances in Environmental Biology
ISSN-1995-0756
EISSN-1998-1066
Journal home page: http://www.aensiweb.com/AEB/
The Role of Azotobacter Chrococcum and Azotobacter Paspali on Wheat Growth in
Fars Province
1Mehdi
Kargar, 2Mohammad Kargar, 2Mohammad Javad Nowrooz Nejad, 3Kavous Ayazpour
Young Researcher’s and Elite Club, Jahrom Branch, Islamic Azad University, Jahrom, Iran.
Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran.
3
Department of Plant Pathology, Jahrom Branch, Islamic Azad University, Jahrom, Iran
1
2
ARTICLE INFO
Article history:
Received 25 June 2014
Received in revised form
8 July 2014
Accepted 25 November 2014
Available online 16 December 2014
Key words:
Azotobacter, nitrogen fixation, animal
manure, wheat
ABSTRACT
Introduction: About 90% of nitrogen fixation is performed by microorganisms.
Azotobacters are important free-living nitrogen-fixing bacteria. In this study the role of
Azotobacter Chrococcum and Azotobacter Paspali in the growth and productivity of
wheat has been investigated. Materials and Methods: The most active Azotobacter
Chrococcum and Azotobacter Paspali in fixation of nitrogen in different wheat fields of
Fars Province were isolated using Winogradskii culture medium. This randomized
study was performed in six replications at outdoors considering the following factors;
1- soil sterile with animal manure and unsterile with animal manure, 2- urea at three
levels (no urea, 100 mg urea per kg soil, and 200 mg urea per kg soil), and 3- bacteria
type at four levels (no bacteria, Azotobacter Paspali alone, Azotobacter Chrococcum
alone, and a mix of both bacteria).Findings: The highest weigh of wheat thousand-seed
was seen in pots treated by Azotobacter Chrococcum and Azotobacter Paspali. Pots
containing unsterile animal manure had the greatest plant growth and in this regard the
difference between various levels of nitrogen fertilizer was significant at 5% level.
Discussion and Conclusion: The region’s indigenous Azotobacter Chrococcum
enhances more wheat growth through nitrogen fixation compared to the other
Azotobacter due to calcareous soil and warm and dry weather of the Province. Organic
compounds such as animal manure degrade gradually and enhance the growth and
stimulate nitrogen fixation by free-living bacteria such as Azotobacters. The indigenous
Azotobacter Chrococcum can also be isolated and used for biologic fertilizers to
improve crop yield.
© 2014 AENSI Publisher All rights reserved.
To Cite This Article: Mehdi Kargar, Mohammad Kargar, Mohammad Javad Nowrooz Nejad, Kavous Ayazpour., The role of Azotobacter
Chrococcum and Azotobacter Paspali on Wheat Growth in Fars Province. Adv. Environ. Biol., 8(17), 837-841, 2014
INTRODUCTION
The main role of soil in the ecosystem is decomposition of organic matter into minerals which are used by
plants. Most microbes in the global ecosystem are present in the soil. Bacteria are the highest and most abundant
group of soil microbes [14]. About 108 bacteria exist in each gram of the most fertile soils [18]. The genus
Azotobacter is a diverse group of free-living microorganisms that have the ability to fix N2 as the sole nitrogen
source [26]. The genus is composed of 7 species, of them Azotobacter chroococcum is found in different soils
[24]. Azotobacter paspali can be found in the rhizosphere of herbs or plants. Soil may contain over thousands of
Azotobacter cells. These bacteria are absent or very low in the acidic pH range [3]. Given their role in the
production of plant growth elements, ability to synthesize antibiotics and vitamins, and anti-pathogenic
properties, they are important in agriculture [15].
Many researchers have tried to find the functional interactions and positive impact of Azotobacter species
on plants and stated that the key factor in the growth is merely their nitrogen-fixing activity. However, the effect
on plant growth and crop production has been demonstrated later [17]. Azotobacters can do this by increasing or
decreasing the absorption of nutrients through plant roots [9]. Organic matter in the soil can enhance the growth
of Azotobacters, but the abundance of organic material can prevent their growth [9,21]. In this study, we
investigated the effect of native Azotobacter chroococcum and Azotobacter paspali, isolated from wheat fields
of Fars Province, as well as the effect of animal manure on the growth of wheat.
Corresponding Author: Mehdi Kargar, Young Researcher’s and Elite Club, Jahrom Branch, Islamic Azad University.
Tel: 09173915663
E-mail: [email protected]
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Mehdi Kargar et al, 2014
Advances in Environmental Biology, 8(17) September 2014, Pages: 837-841
MATERIALS AND METHODS
A total of 50 soil samples were randomly collected from the wheat fields of Fars Province. Isolation was
performed through adding 5 g of soil to 45 mL of nitrogen-free Winogradsky culture medium (the stoke
contained 5 g KH2PO4, 2.5 g MgSO4.7H2O, 2.5 g NaCl, 0.1 g FeSO4.7H2O, and 0.1 g MnSO4.4H2O in 100 mL
distilled water; 5 mL of the stock was poured into 999 mL distilled water and then 0.1 g CaCO 3 and 1 g glucose
was added to it) [23,8]. The Erlenmeyer flask was stirred for 5 minutes to solve soil textures, and then 5 mL of
the culture medium from the first flask was transferred into 45 mL of another Winogradsky culture medium and
diluted to 10-2; each dilution was then cultured on solid Winogradsky and Azotobacter paspali agar media (20 g
sucrose, 0.05 g K2HPO4, 0.15 g KH2PO4, 0.2 g MgSO4.H2O, 0.02 g CaCl2, 1 g CaCO3, 0.002 g Na2MoO4, 0.01
g bromothymol blue, 1 drop 10% FeCl3, and 15 g agar-agar in 1 L distilled water at pH 7) [6,21]. The plates
were incubated at 30 °C for 48 hours [16,3]. After growth on Azotobacter paspali agar and Winogradsky media,
the bacteria were purified and identified through Gram staining, catalase, and biochemical tests, etc. based on
the Azotobacteriace family table and Bergey’s manual [5].
To evaluate the activity of nitrogen fixation, Azotobacters were grown in liquid Winogradsky medium and
incubated at 30 °C for 24 hours, and the production of nitrogenous compounds was evaluated using Nessler,
NO2, and NO3 reagents for a week [5,13].
The soil was sieved, poured into perforated metal dishes, and autoclaved at 121 °C and 15 psi for 15
minutes and then at 180 °C for 2 hours; this eliminated all bacteria in the soil, especially Azotobacters. For
greater certainty, soil was cultured over the surface of Winogradsky medium and no growth of Azotobacters was
observed.
From active Azotobacters, the strains paspali and chroococcum were added to a nitrogen-free Winogradsky
medium. They were then incubated at 30 °C for 24 hours until the turbidity of the culture medium reached to
half McFarland (a concentration in which 1.5108 bacteria exist per mL of culture medium).
The pots were filled with 0.5 kg sterile gravel. Then 4.5 kg limestone soil was poured into each pot and the
wheat seeds (Falat cultivar) were planted in the pots; the pots were then inoculated with 100 mL of the desired
bacteria and irrigated with sterile water. Animal manure was mixed well with the soil before planting. The pots
were placed in the open air and irrigated frequently with sterile distilled water. Six days after cultivation of
wheat, the plants’ rhizosphere in all pots was sampled and diluted with normal saline and then surface cultured
on solid Winogradsky medium. The number of Azotobacter chroococcum and Azotobacter paspali was counted
in each gram of soil and repeated monthly for 4 months. After germinating, the tillers of wheat were counted
and the 1000 seed weight and the soil nitrogen were measured in all pots after 4 months. This study was
performed in a factorial manner with a completely randomized design (CRD) with four factors at outdoor in 5
kg pots with 6 replications. The factors included soil type (sterile with animal manure and non-sterile with
animal manure), urea at three levels (urea-free, 100 mg per kg of soil, and 200 mg per kg of soil), and bacteria
type at four levels (bacteria-free, Azotobacter paspali alone, Azotobacter chroococcum alone, and both species).
The results were statistically analyzed by MSTAT-C and the means were compared through Duncan’s multiple
range test (DMRT).
Result:
When comparing the times of soil sampling, the maximum and the minimum numbers of bacteria were seen
in the third month (2014/03/01) and the first month (01/01/2014), respectively. This is while the difference in
the number of Azotobacter in the pots’ soil was significant between the first and third months and between the
second and third months at the 5% level of Duncan’s test (Chart 1). According to the ANOVA table, a
significant difference was observed between the variable resources of the test at the 1% level. In this regard,
only the difference between various levels of nitrogen fertilizer was significant at the 5% level (Chart 2).
Regarding the interaction of soil and bacteria, the maximum and the minimum number of bacteria were
observed in the sterile soil and fertilizer containing Azotobacter chroococcum and in the bacteria-free normal
soil and fertilizer, respectively. In this regard, no significant difference existed between the sterile and normal
soils and fertilizers without bacteria and with Azotobacter paspaly. But when both bacteria and Azotobacter
chroococcum were used, a significant difference was observed between the sterile and normal soils and
fertilizers (Chart 2).
Both types of soil, containing Azotobacter chroococcum or both types of bacteria, had a higher thousand
seed weight (Chart 3). In terms of the interaction between soil type and nitrogen fertilizer, among 3 levels of
nitrogen fertilizer, the maximum percent of decrease in soil nitrogen was observed in normal soil and in the
sterile soil and fertilizer with 100 mg of urea, and the minimum in the sterile soil and fertilizer with 200 mg of
urea. In this regard, only the difference between the sterile soil and fertilizer containing 200 mg of urea and the
other combinations was significant, but the remaining had no significant difference (Chart 4).
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Advances in Environmental Biology, 8(17) September 2014, Pages: 837-841
Chart 1: The comparison of sampling times in the number of bacteria.
Chart 2: The comparison of mutual impact between soil type and type of bacteria in the number of bacteria.
Chart 3:The comparison of mutual impact between soil type and type of bacteria in seed weight.
Chart 4: The comparison of mutual impact between soil type and nitrogen fertilizer on soil nitrogen levels.
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Mehdi Kargar et al, 2014
Advances in Environmental Biology, 8(17) September 2014, Pages: 837-841
Discussion and Conclusion:
The results showed that the use of non-sterile animal manure with both Azotobacters paspali and
chroococcum yielded the highest 1000 seed weight of wheat. This can be attributed to the simultaneous effects
of the Azotobacters for better plant growth and higher yields (Chart 3). Other studies have also found that
Azotobacters enhance the growth and yields of wheat and other plants [20]. Rajaee et al. [19] showed that
inoculation of Azotobacter chroococcum increases the wheat 1000 seed weight. Zaeid et al. [25] also reported
the positive and significant impact of Azotobacter chroococcum, with the greatest potential to produce indole
acetic acid (IAA), on 1000 seed weight, in the presence of different levels of nitrogen fertilizer. According to
Bahrani et al. [4], Azotobacter chroococcum increases wheat yields. They found that the use of Azotobacter
chroococcum fixes nitrogen in the soil up to 19% and adding small amounts of organic material enhances
nitrogen fixation by bacteria. In addition, Dashadi et al. [7] and Rodelas et al. [22] stated that the use of
Azotobacter chroococcum and Rhizobium leguminosarum increases the yield of bean. In another study by Essam
et al. [10], it was shown that concomitant use of Azotobacter and Azospirillum can fix nitrogen up to 230 kg per
hectare.
The absence of other microbes in sterile soil and animal manure has possibly led to optimized growth of
Azotobacters; in addition, Azotobacter chroococcum was better grown in the calcareous soil, while the growth
of Azotobacters in the non-sterile soil and animal manure was low due to competition of bacteria in soil and
animal manure (Chart 2). Essam et al. [10] suggested that small amounts of organic and inorganic substances
can stimulate greater fixation of nitrogen by bacteria. Also, Gutierrez et al. [11] showed that the addition of
small quantities of organic and inorganic substances stimulates Azotobacter chroococcum and further fixation of
molecular nitrogen. Aquilanti et al. [1] found that the addition of organic matter increases nitrogen fixation by
Azotobacter chroococcum in liquid Winogradsky medium. Simultaneous use of Azotobacters chroococcum and
paspali in this study resulted in nitrogen fixation in wheat, and addition of organic matter, such as animal
manure, further enhanced nitrogen stimulation and fixation by bacteria.
The minimal decline in soil nitrogen was observed in pots with 200 mg urea per kg of soil. Soil nitrogen
loss can be prevented through adding low amounts of urea fertilizer. Regarding the use of sterile and non-sterile
soils and 200 mg urea, a significant relationship existed at p<0.05. Amirhandeh et al. [2] showed that
Azotobacters chroococcum can fix molecular nitrogen up to 45 kg per hectare. Also, Kilzikaya [12] proved that
Azotobacters isolated from soil in Turkey can fix 3.50 to 29.35 µg molecular nitrogen per mL of culture
medium. According to the present research and other studies, the use of native, nitrogen-fixing bacteria can
result in nitrogen fixation and further increase in plant yield.
According to the results, it can be stated that organic materials such as animal manure can enhance the
growth and stimulate nitrogen fixation by free-living bacteria such as Azotobacters. However, soil nitrogen and
Azotobacters growth reduced after three months due to limited resources of soil in the pots. This decline was
compensated in pots with artificially added nitrogen. In addition, the growth of Azotobacter chroococcum was
better than Azotobacter paspali due to calcareous soil of the province.
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