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Khan Mohd. Faraz, Khan Hina; International Journal of Advance Research, Ideas and Innovations in Technology
© 2019, www.IJARIIT.com All Rights Reserved Page |90
ISSN: 2454-132X
Impact factor: 4.295
(Volume 5, Issue 5)
Available online at: www.ijariit.com
Isolation of bacteria from agricultural soil and screening it for
PGPR traits
Mohd. Faraz Khan
khanfraz2341@gmail.com
Indian Institute of Technology, Indian School of Mines,
Dhanbad, Jharkhand
Hina Khan
khan.hina0017@gmail.com
Bureau of Indian Standards, New Delhi,
Delhi
ABSTRACT
Rhizobacteria owning multiple plant growth-promoting activities were isolated from the rhizospheric soils of plants flourishing
in a semi-arid region. Plant Growth Promoting Rhizobacterial (PGPR) strains were segregated and screened for their plant
growth-promoting activities like phosphate solubilization, production of indole- acetic acid, ammonia, hydrogen cyanide
(HCN). Bacteria that colonize plant roots and promote plant growth are referred to as plant growth-promoting rhizobacteria
(PGPR). PGPR is highly assorted and in this review, we focus on rhizobacteria as biocontrol agents. PGPR can affect growth
directly or indirectly. Direct promotion of plant growth by PGPR involves both providing plants with a compound synthesized
by the bacterium or helping the uptake of certain nutrients from the environment; while mechanisms of biological control by
which rhizobacteria can support plant growth indirectly, i.e., by decreasing the level of disease, include antibiosis, induction of
systemic resistance, and struggle for nutrients and niches.
Keywords— PGPR, Nutrients, Biological fertilizers, Phosphate, Solubilization
1. INTRODUCTION
Quality and quantity of food are going to be important challenges in coming time Continuous population growth requires
production of more agricultural products and to inevitably move towards increased production per unit area. This cannot be
achieved without the application of either chemical or bio-based fertilizers. Since fertilizer management is considered as one of
the main factors of sustainable agriculture, gradual replacement of chemical fertilizers with biological fertilizers is quite inevitable
due to their advantages and cost-effectiveness. The history of plant inoculation with useful bacteria goes back to many centuries
ago. For instance, by experience, farmers knew that if the soil in which legumes were planted was mixed with the soil for non-
legume crops, it resulted in an increased crop yield. In late 19th century, the first license for producing a biological fertilizer
known as Nitragin was issued for the production of rhizobium inoculants and after that, inoculation of legumes started to be
practiced in many countries using rhizobium fertilizers [1]. The rhizosphere, the narrow zone of soil that surrounds and influences
the plant roots, is home to a large number of microorganisms and is considered to be one that can have profound effects on the
growth, nutrition and health of plants in agro-ecosystems [2]. The rhizosphere, microbiota can contain up to 1011 microbial cells
per gram of root [3] and more than 30,000 prokaryotic species [4]. Bacteria able to colonize plant root systems and promote plant
growth are referred to as plant Growth Promoting Rhizobacteria (PGPR) [5].
Fig. 1: Representation of a Rhizospheric Zone [6]
Khan Mohd. Faraz, Khan Hina; International Journal of Advance Research, Ideas and Innovations in Technology
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Table 1: PGPR and their effect on growth parameters/ yields of crop/fruit plants
1.1 Location of study area
Unnao district represents flat topography with a general elevation of 98 m (322 ft.) covering an area of 4558 km2 . By virtue of its
geographic setting in the great (Ganga) plains, the land is highly fertile. The soil is mostly alluvial. The district is mainly drained
by the river Ganga and its tributaries Kalyani, Khar, Loni and Marahai in the western part of the district and by Sai river in the
eastern part of the district. All these rivers are perennial in nature. About 87% area of the net sown area (3,00,000 hectares) is
irrigated both by surface water (Sharda Canal network system) and ground water through shallow and moderately deep tubewells.
The share of surface water irrigation is 48% while that of ground water is 52%.
Soil found in Unnao industrial and surroundings village of Unnao district exhibit a wide variance in composition and appearance.
The major part of area consists of ordinary soils known locally as Bhur or sand on the ridges, Matiar or clay in the topographic
lows and Dumat or loam on the plains. Clay is dominant in the areas where "Reh" or USAR prevails. Alluvial soils of river valleys
notable the "Kachhar" of the Ganga formed by repeated deposition of silt brought down by the existing river system during floods.
[7]
Fig. 2: Satellite Imagery of the Soil Sampling Location
Khan Mohd. Faraz, Khan Hina; International Journal of Advance Research, Ideas and Innovations in Technology
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2. MATERIAL AND METHOD
2.1 Sample Selection
The plant growth promoting rhizobacteria were isolated from the rhizosphere of following plants from the agricultural soils of
Unnao Region:
2.1.1 Common wheat (Triticum aestivum): Loosely attached soil was separated from the roots. The roots were shaken tenderly
to remove excess soil. After extracting the soil loosely adhering to root, the soil adhering firmly to the root of each plant was
accumulated through brushing (termed rhizosphere soil sample, RSS).
2.2 Preparation of Nutrient Broth
Nutrient Broth is used for the general cultivation of less fastidious microorganisms, can be enriched with blood or other biological
fluids. Table 2: Composition of Nutrient broth Media
S no.
Ingredients
g/L
1
Peptic digest of animal tissue
5.00
2
Sodium chloride
5.00
3
Beef extract
1.50
4
Yeast extract
1.50
5
Final pH ( at 25°C)
7.1±0.2
2.3 Serial Dilution for isolation of Bacteria from the soil
The Purpose of serial dilution was to determine the number of bacteria per unit volume in the original culture, determination of the
culture density in cells per ml. Once the culture had been diluted it could be spread on agar plates. Agar plates allow for individual
bacterial cells to be separated spatially. If done correctly, there is a low probability of having two cells very close to each other.
When each of these spatially separated cells multiplies, spatially separated colonies were formed.
2.3.1 Procedure: The soil samples were serially diluted from 10-3 to 10-7 dilutions using sterile distilled water as a blank and
they were inoculated on the nutrient agar medium by pour plate technique. After 24 hours of incubation at 37°C the colonies were
counted.
Fig. 3: Pour Plate Method
2.4 Streak Plate Method
2.4.1 Streak plate technique: is used for the isolation into pure culture of the organisms (mostly bacteria), from mixed
population. The inoculum is streaked over the agar surface in such a way that it “thins out” the bacteria. Some individual bacterial
cells are separated and well spaced from each other.
2.4.2 Procedure: This technique was done using an inoculating loop. 0.1 mL of the bacterial suspension was placed in the center
of the plate using it.The glass rod was sterilized by first dipping it into a 70% alcohol solution and then passing it quickly through
the Bunsen burner flame. The burning alcohol sterilizes the loop at a cooler temperature than holding the rod in the burner flame
thus reducing the chance of burning fingers.
Fig. 4: Performing Serial Dilution of Bacteria from the Soil (Isolation) and Performing the Streak Plate Method in
Laminar Air Flow Chamber
Khan Mohd. Faraz, Khan Hina; International Journal of Advance Research, Ideas and Innovations in Technology
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Screening of Phosphate Solubilizing Bacteria (PSB) .In most bacteria, mineral phosphate-dissolving capacity had been shown to
be related to the production of organic acid such as Gluconic Acid (GA) [8], earlier which was reported to produce by direct
oxidation of glucose. Mainly the biosynthesis of GA reported to carry out by the Glucose Dehydrogenase (GDH) enzyme and the
co-factor, Pyrroloquinoline Quinone (PQQ). The p-sol ability was checked on Pikovskaya’s media plate. Mainly this media
contains calcium phosphate which acts as a source of phosphate. Moreover, GA producing rhizobacteria could be easily
differentiated on the Pikovskaya’s media plate by observing a clear halo zone due to the release of phosphorous from the media.
Following steps were employed for isolation of PSB:
(a) 1 g dried Rhizospheric Soil Sample (RSS) was added to a 25 ml flask with 9.0 ml sterilized distilled water and incubated for
30 minutes (min) at 30 °C in a shaker at 250 RPM. The resulting suspension was decimally diluted (10‾²-10‾⁶) with sterilized
distilled water.
(b) All RSS were appropriately diluted and plated on Pikovskaya agar media to get approximately 100 colonies per plate. The
plates were incubated at 30 °C for 2 days. Colonies that showed halo zone was considered as PSB.
(c) Reconfirmation of PSB was done on Pikovskaya’s media plates. (d) All PSB strains were streaked on nutrient agar media for
colony purification.
(d) Purified strains were re-analyzed for p-sol on Pikovskaya’s media plates.
(e) Glycerol stocks of all P-solubilizing strains were made in 30% glycerol and stored at -80 °C freezer.
2.5 Phosphate solubilization
Bacterial culture was spot-inoculated on the surface of the plate containing Pikovaskaya’s medium and incubated in an incubator
at 28 °C for 7 days. P-solubilization was determined by the development of the clearing zone around a bacterial colony [9]
2.6 Production of Ammonia
Bacterial isolate was tested for the production of ammonia in peptone water. The freshly grown culture was inoculated in 10 ml
peptone water in a test tube and incubated for 48-72 h at 36±2 °C. Nessler’s reagent (0.5 ml) was added to each tube.
Development of brown to the yellow color indicated a positive test for ammonia production [10].
2.7 Production of Indole acetic acid
Bacterial culture was grown in LB medium amended with 100 mg L-1 tryptophan as the precursor of IAA by incubating in a
shaker at 250 rpm at 28 ± 2oC for 7 days. Indole acetic acid (IAA) production was assayed colorimetrically by using Salkowski
reagent (1ml of 0.5M FeCl3 in 50 ml of 35% HClO4) and the absorbance of the resultant pink color at 535nm in the colorimeter.
The appearance of a pink color in test tubes indicated IAA production. The concentration of IAA was determined by comparison
with a standard curve [11].
2.8 HCN Production
Briefly, the strain was streaked on the nutrient agar amended with glycine (4.4 g/L). A Whatman filter paper No.1 soaked in 2%
sodium carbonate prepared in 0.5% picric acid solution was placed at the top of the plate. The plated sealed with a parafilm were
incubated at 28±2oC for 4 days change of filter paper from orange to red if found, was noted down, hence confirming HCN
production.[12]
Table 3:
Examples of different phytohormone-producing PGPR
3. RESULT AND DISCUSSION
Table 4: Plant Growth Promoting Traits of the Isolate
PGP Traits
Response
Ammonia Production
+ve
Phosphate Solubilization
+ve
IAA Production
-ve
HCN
+ve
3.1 Phosphate Solubilization
On Pikovaskaya’s agar plates a clear zone was observed around the bacterial colonies. The solubilization ability of rhizosphere
microorganisms is considered to be one of the most important traits associated with plant phosphate nutrition. It is generally
accepted that the mechanism of mineral phosphate solubilization by PSB strains is associated with the release of low molecular
weight organic acids, through which their hydroxyl and carboxyl groups chelate the cations bound to phosphate, thereby
converting it into soluble forms. PSB has been introduced to the Agricultural community as phosphate Bio fertilizer.
Khan Mohd. Faraz, Khan Hina; International Journal of Advance Research, Ideas and Innovations in Technology
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Fig. 5: Phosphate solubilization by the isolates
3.2 HCN Production
A change in color of the filter paper was noticed from light orange to reddish orange, which confirmed the production of HCN by
the isolated bacterial strain. Hence the isolating strain might be PGPR. The production of the HCN by the isolates is a healthy sign
that the plant will resist activities in the rhizosphere or root zone due to the pathogenic bacteria or fungal activity.
3.3 NH3 Production
In addition, of Nessler’s reagent to put-on water inoculated with bacterial strain, a considerable change in color from brown to
yellow was noticed, thus confirming the production of ammonia. There are a number of sources of ammonia secretion of
rhizospheric microorganisms. Ammonia and extracellular proteins are the nitrogenous secretions of nitrogen fixers in nitrogen free
or deficient medium.
Fig. 6: HCN production by the isolates (orange color)
Fig. 7: Ammonia production by the isolates (yellow colour)
4. CONCLUSION AND RECOMMENDATIONS
PGPR are found in plant roots or in the adjacent soil and contribute to the plant’s growth and development through multiple direct
and indirect mechanisms. PGPR have been investigated in search of efficient ways to use them to improve agricultural production
in a low impact ecological way. All the isolated strains in this study showed varying levels of plant growth promoting activities,
and all had an overwhelmingly positive effect on the plants for all the investigated bioprocesses: IAA production, Nitrogen
fixation and cellulase activity in addition to their antifungal properties. Based on these results, the isolated PGPRs in this study
could constitute an efficient and more eco-friendly alternative to chemical fertilizers and fungicides in the processes of
biostimulation, bio-fertilization and biological control.
5. REFERENCES
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[2] Berendsen R, Pieterse C, Bakker, P. The rhizosphere microbiome and plant health. Trends in Plant Sciences. 2012; 17:478-
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[3] Egamberdieva D, Kamilova F, Validov S, Gafurova L, Kucharova Z, Lugtenberg B. High incidence of plant growth-
stimulating bacteria associated with the rhizosphere of wheat grown on salinated soil in Uzbekistan. Environmental
[4] Mendes R, Kruijt M, de Bruijn I, Dekkers E, van der Voort M, Schneider JH, Piceno YM, DeSantis TZ, Andersen GL,
Bakker PA, Raaijmakers JM. Deciphering the rhizosphere microbiome for disease-suppressive bacteria. Science. 2011;
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[5] Kloepper JW, Schroth MN. Plant growth promoting rhizobacteria on radishes. 4th International Conference Plant Pathogenic
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[6] Saleem, Muhammad & Pervaiz, Zahida & Traw, Milton. (2015). Theories, Mechanisms and Patterns of Microbiome Species
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[7] CGWB (2010), Ground Water Contamination in Industrial Area, Unnao District, Uttar Pradesh, Central Ground Water Board,
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[8] Rodriguez H, Fraga R. Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnology Advances.
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[9] King JE. The colorimetric determination of phosphorus. Biochemical Journal. 1932; 26:292-97.
[10] Cappuccino J. C. and Sherman N. (1992). In: Microbiology: A Laboratory Manual, third ed. Benjamin/cummings Pub. Co.
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[11] Methods for Growing Spirillum lipoferum and for Counting It in Pure Culture and in Association with Plants
[12] Lorck, H. 1948. Production of hydrocyanic acid by bacteria. Physiol. Plant. 1: 142-146
