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DOI: 10.4172/2332-0737.1000132

Systems Biology
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ISSN: 2332-0737

Research
Research Article
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Isolation and Characterization of Plant Growth Promoting Rhizobacteria

Associated with Saccharum officinarum L.
Krunal Modi1* and Prittesh Patel2
Aspee Shakilam Agricultural Biotechnology Institute, Navsari Agricultural University, Surat, Gujarat, India
1

CG Bhakta Institute of Biotechnology, Uka Tarsadia University, Bardoli, 394350, Gujarat, India
2

Abstract
Plant growth promoting rhizobacteria is free living soil microorganisms that directly or indirectly exert beneficial
effects on plants. Hundred strains were characterized morphologically and biochemically. Primary screening was
carried out for all hundred isolates for IAA, phosphate solubilization, siderophore and nitrogen fixation. Eight isolates
F181, F271, F323, F372, F373, F531, ELB1 and ESB4 from different sites and sugarcane cultivars have showed
potential for all PGPR activities and were further investigated quantitatively. The isolates could exhibit more than two
or three PGP traits, which may promote plant growth directly or indirectly or synergistically. The results shown, F372
achieved the highest phosphate solubilization of 15 mg L-1, and F271 was the most promising IAA producer with 63 mg
L-1. Furthermore, most of the PGPR isolates shown antifungal properties like HCN chitinase and protease production.
The present study, therefore, suggests that the use of selected PGPR isolates as inoculants might be beneficial for
sugarcane cultivation.

Keywords: Bio-control; Rhizobacteria; Phosphate solubilization; solubilization, Nitrogen fixation, Siderophore production, anti-fungal
Siderophore; Sugarcane activity, protease activity, HCN production, cellulase activity, chitinase
activity.
Introduction
Material and Methods
Sugarcane (Saccharum officinarum L.) is a monocotyledon and
belongs to the tribe Andropogoneae of the family Poaceae. It grows Sampling sites, isolation and characterization
best under warm conditions with high light intensity and fertile soil. Soil samples were collected from Sugarcane crop rhizosphere from
Sugarcane differs from many common grasses in its long period of
different villages of Bardoli region includes Sevani, Vansdarundhi,
vegetative growth, resulting in the large size and they have stout,
Vihan and Dhamdod. Sugarcane leaves and stems were collected from
jointed, fibrous stalks that are rich in sugar. It is common in tropical and
a 4 month old plantation. Isolation was carried out by following two
subtropical countries throughout the world. It is one of the principle
crops of South Gujarat and Saurashtra region of Gujarat state. Importance ways. (a) Soil sample was diluted and plated on nutrient agar plates.
of sugar in human diet needs no introduction; it has become a part and Colonies were randomly selected on the basis of morphology and
parcel of daily life. As sugarcane is a long duration crop and faces many further purified by streaking [6]. (b) The leaves and stem were washed
biotic and abiotic stresses during growth phase, the microorganism macerated separately and dilutions were placed on nutrient agar.
associated with sugarcane roots may be potent and useful for application Morphology and texture of each colony was recorded. Colonies were
to promote plant growth [1]. Soil microorganisms play a significant role randomly selected and further purified and maintained on nutrient
in regulating the dynamics of organic matter, decomposition and the agar slant at 4°C for further studies [7].
availability of plant nutrients such as Nitrogen (N), Phosphorous (P)
and Sulfur (S). It is well-recognized that microbial inoculants constitute Phosphate solubilization
an important component of integrated nutrient management that Phosphorus is an essential element next to nitrogen influencing
leads to sustainable agriculture. In addition, microbial inoculants can the plant growth and production. It is absorbed as various organic
be used as an economic input to increase crop productivity; fertilizer and inorganic forms in the plant. The Pikovskya’s medium plates were
doses can be lowered and more nutrients can be harvested from the soil inoculated with isolates then examined for formation of halo zone
[2]. Biofertilizer are the formulation of living microorganisms, which around the colony [8]. The quantitative phosphate solubilization by
are able to fix atmospheric nitrogen in the available form for plants
bacterial isolates was measured using Reyes basal medium [9]. The blue
either by living freely in the soil or being associated symbiotically with
color intensity of the solution was measured at 600 nm.
plants. Biofertilizer are inputs containing microorganisms which are
capable of mobilizing nutritive elements from nonusable form to usable
form through biological processes [3]. A number of bacterial species
associated with the plant rhizosphere belonging to genera Azospirillum, *Corresponding author: Krunal Modi, Aspee Shakilam Agricultural Biotechnology
Institute, Navsari Agricultural University, Surat, Gujarat, India, Tel: 02637 293 804,
Alcaligenes, Arthrobacter, Acinetobacter, Bacillus, Burkholderia, E-mail: [email protected]
Enterobacter, Erwinia, Flavobacterium, Pseudomonas, Rhizobium and
Serratia are able to exert a beneficial effect on plant growth [4]. Plants Received June 15, 2017; Accepted June 30, 2017; Published July 06, 2017
play an important role in selecting and enriching the types of bacteria Citation: Modi K, Patel P (2017) Isolation and Characterization of Plant Growth
by the constituents of their root exudates. Rhizospheric bacterial Promoting Rhizobacteria Associated with Saccharum officinarum L. Curr Synthetic Sys
Biol 5: 132. doi:10.4172/2332-0737.1000132
communities have efficient systems for uptake and catabolism of
organic compounds present in root exudates [5]. We are trying here to Copyright: © 2017 Modi K, et al. This is an open-access article distributed under
isolate the PGPR from sugarcane having potential to act as biofertilizer the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
with the objectives such as screening of IAA production, Phosphate source are credited.

Curr Synthetic Sys Biol, an open access journal Volume 5 • Issue 1 • 1000132
ISSN: 2332-0737
Citation: Modi K, Patel P (2017) Isolation and Characterization of Plant Growth Promoting Rhizobacteria Associated with Saccharum officinarum L. Curr
Synthetic Sys Biol 5: 132. doi:10.4172/2332-0737.1000132

Page 2 of 4

IAA production has been involved in suppression of soil borne pathogens [17]. HCN
is produced by many rhizobacteria and is postulated to play a role in
The production of Indole acetic acid (IAA) was assayed by using
biological control of pathogens. Hydrogen cyanide production was
Salkowski method [10]. Productions of pink colour indicate presence
assayed by the method suggested by Lorck [18] and Castric [19].
of IAA in the medium. And then light absorbance was measured
Discoloration of the filter paper form yellow to brown indicates
immediately at 535 nm spectrometrically. The amount of IAA produced
production of cyanide.
was calculated using the standard curve prepared with known
concentration of IAA. Chitinase production
Siderophore production Chitin is the second most abundant natural polymer and widely
Siderophore is an important for the survival and growth of bacteria distributed as a structural component of crustaceans, insects, and other
in the soil and aqueous environment [11]. Siderophore production was arthropods, as well as a component of the cell walls of most fungi and
tested qualitatively using chrome azurole S (CAS). Orange halos around some algae. For chitinolytic activity bacterial isolates were streaked
the colonies after 48 hours incubation indicated siderophore production. on chitin agar medium. Isolates were detected by the appearance of
Quantitative estimation of siderophores was done by CAS-shuttele assy. clear zones around colonies grown on chitin agar plates. Chitinase
All positive cultures were compared for siderophore production ability activity was measured with colloidal chitin as a substrate. The amount
as described by Payne [12] where percent decolorization was calculated of reducing sugar produced was measured by the dinitrosalicylic acid
by using the following formula: (DNSA) method.

% Siderophore Units = (Ar-As /Ar) × 100 Results and Discussion

Where Ar=Absorbance of reference at 630 nm (CAS reagent) and Isolation and characterization of rhizobacteria
As=Absorbance of sample at 630 nm.
In this study total 100 isolates were isolated from 45 samples
Nitrogen fixation collected from different villages of Bardoli region (Table 1).
During the biological nitrogen fixation, molecular nitrogen is Among all sites, F4 which contained Co 86032 sugarcane variety
reduced in multiple electron transfer reactions, resulting in the synthesis was found good for colonizing higher number of rhizospheric bacteria,
of ammonia and the release of hydrogen [13]. Ammonium is then used while F2 site where sugarcane variety Co-0411 was found poor for
for the subsequent synthesis of biomolecules. Screening of nitrogen fixing colonization of rhizospheric bacteria. In addition leaves and stem of
organisms was carried out by using semisolid malate medium (NFB). Growth Co 86249 were used for isolation of bacteria. Among the total isolates,
of bacterial isolates indicates nitrogen fixation [14]. 59 isolates were found as IAA producer, 61 isolates were found as
Protease production phosphate solubilizes, 39 isolates were found as siderophore producer,
59 isolates were found as nitrogen fixer and 65 isolates were found as
Protease is a single class of enzyme used in detergents, protease producer. The results of primary screening are summarized in
pharmaceuticals, leather, and the food agriculture industries [15]. Table 2.
It constitutes over 40% to 60% of the enzyme industries. Protease
production was assayed using skim milk agar. Proteolytic activities After initial screening for all four PGPR activities, F181, F271, F323,
were identified by clear zone formation around the cell. The enzyme F372, F373, F531, ELB1 and ESB4 were selected for further studies
activity was determined by using McDonald and Chen method [16]. as they have shown positive result. Microscopic observations were
After the completion of the time the absorbance was read out at 700 nm performed to investigate the Gram reaction of PGPR isolates. Effort
spectrophotometrically. was made to characterize all 8 isolate morphologically (Table 3). Two
isolates F181 and ESB4 were Gram positive while rests of the isolates
Cyanide production were Gram negative bacteria. All the isolates were found to having a
Production of HCN by certain strains of Pseudomonas fluorescence rod shape, small in size. Various biochemical tests were performed to

Sample site Source No. of sample collected Sugarcane variety Sampling site No. of isolates obtained
F1 Rhizosphere 08 Co 86032 Sevani 16
F2 Rhizosphere 08 Co 0411 Vansdarundhi 14
F3 Rhizosphere 08 Co 86249 Vihan 21
F4 Rhizosphere 08 Co 86032 Dhamdod 23
F5 Rhizosphere 08 Co 0411 Bardoli 18
F6 Leaves & Stem 05 Co 86249 Vihan 08
Table 1: Details of isolates collected from sugarcane.

Total Isolates
Sample site
IAA production Phosphate solubilization Sideroph-ore production Nitrogen fixation
F1 07 12 04 07
F2 09 11 05 04
F3 12 16 06 14
F4 10 05 07 10
F5 17 12 10 15
F6 04 05 07 07
Table 2: PGPR activities among the total isolates.

Curr Synthetic Sys Biol, an open access journal

Volume 5 • Issue 1 • 1000132
ISSN: 2332-0737
Citation: Modi K, Patel P (2017) Isolation and Characterization of Plant Growth Promoting Rhizobacteria Associated with Saccharum officinarum L. Curr
Synthetic Sys Biol 5: 132. doi:10.4172/2332-0737.1000132

Page 3 of 4

Selected Isolates
Characteristics
F181 F271 F323 F372 F373 F531 ELB1 ESB4
Grams nature Positive Negative Negative Negative Negative Negative Negative Positive
Size Small Large Small Small Small Small Small Small
Shape Round Round Round Round Round Round Round Round
Margin Entire Entire Entire Entire Entire Entire Entire Entire
Opacity Transleucent Opaque Opaque Opaque Opaque Transparent Transparent Opaque
Elevation Raised Convex Flat Flat Flat Raised Convex Convex
Consistency Moist Dry Moist Moist Moist Moist Moist Moist
Colour Yellow Green Colorless Orange Green Orange Colorless Colorless
Table 3: Morphological characterization of selected isolates.

Selected Isolates
Characteristics
F181 F271 F323 F372 F373 F531 ELB1 ESB4
Glucose + + + + + + + +
Maltose + + + + - - - -
Lactose - + + + + - - +
Mannitol + - + + - - + +
Xylose - + - - + - - -
Sucrose - - + + - - + +
Methyl Red Test - + - - - - - -
Voges-Proskauer + - + + + + + +
Oxidase test - + + - -
Citrate Utilization + + + + + + - +
Urea Hydrolysis + - - - - - - -
H2S Production - - - + - - + -
Phenyl alanine - - + - - - - -
Nitrate Reduction + + + + + + + +
Ammonia Production + - + - - - - -
Gelatin Hydrolysis + + + - - - + +
Catalase + + + + + + + +
T Gas + - - - - - - -
S H2S - + + - - - + -
I Lac fermentor - + + - - - - -
Table 4: Biochemical characterization of selected isolates.

Isolates Phosphate (mg L-1) IAA (mg L-1) Siderophore Protease (IU) Chitinase (IU)
F181 10 56.23 92 19.5 0.35
F271 6 63.69 72 5.41 0.2
F323 8 38.48 91 7.58 -
F372 15 12.43 94 12.78 -
F373 12 31.91 95 15.81 -
F531 9 36.18 95 11.7 -
ELB1 7 49.66 96 17.3 -
ESB4 14 29.49 88 13.43 -
Table 5: Quantitative estimation of PGPR properties.

identify the isolate including sugar fermentation, oxidase test, urea, TSI development processes. As a critical plant hormone, auxin modulates
etc. (Table 4). such diverse processes as tropic responses to light and gravity, general
root and shoots architecture, organ patterning, vascular development
Phosphorus is second highest required mineral nutrient after and growth in tissue culture [24]. IAA production was found 93.69 mg
nitrogen in the growth of plants. Phosphate solubilising rhizobacteria L-1 by F271 isolate which was highest in all isolates being analyzed. F372
have been considered as one of the best alternatives for inorganic isolate was found poor in IAA production. F181, F271 and ELB1 found
phosphate fertilizers for promoting plant growth and yield [20-22]. to produce more than 40 mg L-1. All eight organisms were grown well
Out of 61 bacterial isolates, eight isolates shown prominent phosphate on N free media which indicates that isolates are able to fix maximum
solubilization were further evaluated quantitatively. Among all eight nitrogen. A plant is better able to achieve its optimized physical growth
isolates F372 (15 mg L-1) was found best isolates as phosphate solubilizers when it receives enough nutrients such as fixed nitrogen and this can be
followed by ESB4 (14 mg L-1) (Table 5). Similar consistent results of influenced by the presence of such bacteria in association with the host
phosphate solubilization in both agar and broth assay were observed plants [25]. These biological processes can help reduce overreliance
earlier [1,23]. Quantification of IAA was carried out for eight different on chemical fertilizer. Siderophore by PGPR is one of the biocontrol
isolates. The phytohormone regulates a whole repertoire of plant mechanisms under iron limiting condition. PGPR produces a range

Curr Synthetic Sys Biol, an open access journal

Volume 5 • Issue 1 • 1000132
ISSN: 2332-0737
Citation: Modi K, Patel P (2017) Isolation and Characterization of Plant Growth Promoting Rhizobacteria Associated with Saccharum officinarum L. Curr
Synthetic Sys Biol 5: 132. doi:10.4172/2332-0737.1000132

Page 4 of 4

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