Int.J.Curr.Microbiol.App.Sci (2013) 2(10): 406-417
ISSN: 2319-7706 Volume 2 Number 10 (2013) pp. 406-417
http://www.ijcmas.com
Original Research Article
Characterization of Bacillus sp. strains isolated from rhizosphere of tomato
plants (Lycopersicon esculentum) for their use as potential plant growth
promoting rhizobacteria
D.Pavan Kumar Agrawal1 and Shruti Agrawal2*
1
Department of Biotechnology, G. B Pant Engineering Collge, Ghurdauri, Pauri,
Uttrakhand, India
2
Department of Microbiology, Sai institute of paramedical and allied Sciences,
26/26A, Rajpur road, Near Meedo grand hotel, Dehradun-248001, India
*Corresponding author
ABSTRACT
Keywords
Plant growth
promoting
rhizobacteria;
IAA
production;
phosphate
solubilization;
Seed
germinationl
roll towel
method.
Plant growth promoting rhizobacteria (PGPR) influence the plant growth by
various direct or indirect mechanisms. To look for efficient PGPR strains having
multiple activities, total 28 bacterial isolates were isolated from different
rhizospheric soil of tomato crop in the vicinity of Dehradun. The bacterial isolates
were biochemically characterized and screened in vitro for their plant growth
promoting traits like production of indole acetic acid (IAA), phosphate
solubilization, hydrogen cyanide (HCN), siderophore and catalase. Total 5 bacterial
isolates of Bacillus showed potential PGPR activities. The productions of indole
acetic acid (IAA) by all rhizobacteria were investigated as important mechanism
for plant growth stimulation. All rhizobacterial isolates produced IAA in vitro by
the addition of L-tryptophan, in the culture medium. All rhizobacterial isolates in
comparison with the control, also showed increment in both the shoot and the roots
of tomato seedlings. PGPR were tested for seed germination and seedling vigour by
using tomato seeds in roll towel method. Generally, the seed germination ranged
from 90 percent (uninoculated control) to 97.5 percent. All the strains, HBS-VIII,
FAR-IIIb, HBR-II, GAR-III and HBR-VII significantly improved seed germination
when compared to the uninoculated control (UIC). The isolate HBRVII showed
significantly increased seed germination (97.5%) and also shoot and root length as
well as enhanced vigour index of 115.50, 714.35 after 6 and 16 days respectively.
The rhizobacteria could exhibit more than two or three PGPR traits that promote
plant growth directly or indirectly.
Introduction
Microbial communities are abundantly
found in the rhizosphere and also in the
area that is under the influence of the root
and its close vicinity. The rhizosphere
406
gives support to many active microbial
populations capable of exerting beneficial,
neutral or detrimental effects on plant
growth (Latour et al., 1996). In this paper
Int.J.Curr.Microbiol.App.Sci (2013) 2(10): 406-417
we focus on the bacteria that are derived
from root and exert benificial effect on the
root. Such bacteria are generally
designated as plant-growth-promoting
rhizobacteria (PGPR). PGPR are a
heterogeneous group of bacteria that are
found in the rhizosphere, at root surfaces
and in association with roots, which can
improve the extent or quality of plant
growth directly and/or indirectly. Direct
effects depends on the production of plant
growth regulators, improvement in plant
nutrients uptake, or promote induce
systemic resistance (ISR) of the plant
(Chaudhary 2011).
It has seen that various species of Bacillus
are of interest as they are dominant in soil
and are known to secrete antimicrobials
and siderophores. In addition, they are
also important microbial producers of
biosurfactants such as rhamnolipis. These
can cause lysis of zoospores of soil borne
plant pathogenic fungi such as Pythium,
Phytophthora and Plasmopara by
interacting with and disrupting their
plasma membrane (Stanghellini and
Miller, 1997). Consequently, Bacillus and
other rhizobacteria can facilitate a control
of damping-off especially in vegetable
nurseries where the disease is often
prevalent by destroying zoospores of these
phytopathogen. Interestingly, protection
against diseases by rhizobacteria also
involves quorum sensing in rhizosphere
(Gray and Smith, 2005, Sharma et al.,
2003) and induction of systemic resistance
(ISR) wherein lipopolysaccharides have
been reported to act as signal molecules
(Pathak et al., 2004).
The indirect promotion of plant growth
occurs when PGPR prevent deleterious
effect. The exact mechanisms by which
PGPR promote plant growth are not fully
understood, but are thought to include (i)
the ability to produce or change the
concentration of plant growth regulators
like indole acetic acid (Patten and Glick,
2002), gibberellic acid , cytokinins and the
ability to produce ACC deaminase to
reduce the level of ethylene in the roots of
the developing plants, thereby increasing
the root length and growth (4. Glick,1995),
(ii) asymbiotic N2 fixation (Kennedy et al.,
1997),
(iii)
antagonism
against
phytopathogenic
microorganisms
by
producing siderophores, b-1,3-glucanase,
chitinases, antibiotics, fluorescent pigment
and cyanide (6. Sharma et al 2003) (iv)
solubilization of mineral phosphates and
other nutrients (Johri et al., 2003).
Currently our research effort is towards
helping the poor farmer communities,
particularly those in the state of
Uttarkhand in the Central Himalayan
region, that depend only on vegetable
cultivation for their livelihood. In this
study we focus on role the of rhizobacteria
in plant growth promotion. A pool of
promising rhizobacteria was screened
through in-vitro and their plant growth
promoting properties. To evaluate the
influence of the most promising bacterial
strains on plant growth, bacterized tomato
seed were planted in paper towel method.
The potential of these has been
analyzed,that is to provide data that effect
the growth parameters in tomato.
In last few decades a large array of
bacteria
including
species
of
Pseudomonas, Azospirillum, Azotobacter,
Klebsiella, Enterobacter, Alcaligenes,
Arthrobacter, Burkholderia, Bacillus and
Serratia have reported to enhance plant
growth (Kloepper et al., 1989; Glick,
1995).
Consequently,
Uttarakhand
region
represents a unique combination of plant
and soil type which changes drastically
407
Int.J.Curr.Microbiol.App.Sci (2013) 2(10): 406-417
with altitude. However limited efforts
have been done so far to explore the
available bacterial diversity. In the present
study rhizospheric soil samples from
tomato were collected from Dehradun
region for assessment of PGPR. The
objectives of this study is to isolate and
characterize Bacillus sp. from the
rhizosphere of tomato, and to screen their
abilities and the possession of direct and
indirect
plant
growth
promoting
rhizobacteria attributes.
rhizosphere. The remaining bacterial
isolates were phenotypically (morphotypic
and physiological) characterized. Colony
morphology of isolates was studied under
a stereoscope microscope (Leica). This
included shape, edge, elevation, surface
and pigmentation.
The cellular morphology was based on cell
shape and Gram staining (Leica
fluorescent microscope). The Bacterial
identification was carried out on the basis
on Bergeys Manual of Systematic
Bacteriology (Clauss and Berkeley, 1986).
The Bacillus species were estimated on the
basis of morphological and physiological
characteristics.
Materials and Methods
Isolation of Bacillus from the
rhizosphere
Characterization of rhizobacteria for
PGP traits
Rhizospheric soil samples were collected
from tomato growing fields of Dehradun.
Bacillus species were isolated using
dilution method with Nutrient Agar
medium. Rhizosphere soil samples (10 g)
were suspended in 90 mL of 0.85%
normal saline (pH 7.0) and shaken
vigorously at 150 rpm at 18°C for 1 h. The
resulting slurry was serially diluted (100
L) to 900 L of 0.85% normal saline in
each Eppendorf tube and appropriate
dilution (10-4) of this suspension (0.1 mL
or 100 L) was spread plated in triplicate
on Nutrient Agar medium. Cultures were
incubated at 37°C±2 for 2 d. For
experimental use, isolates were transferred
when needed to nutrient agar medium that
was stored at 4°C. Each colony was
assayed further for morphological and
physiological characteristics including
Gram reaction, endospore, motility,
oxidase and catalase enzyme activity.
Siderophore production
Siderophore production was tested
qualitatively using chromeazurol S
medium (CAS-medium) ( Sharma and
Johri, 2003). Each Bacillus isolate were
streaked on the surface of CAS agar
medium and incubated at room
temperature for 1 to 3 days. Siderophore
production was confirmed by orange halos
around the colonies after the incubation,
and this test was done in two replications.
Phosphate solubilization
Solubilization of tri-calcium phosphate
was detected in Pikovskaya s Agar ( Johri
et al., 2003). All bacterial isolates were
streaked on the surface of Pikovskaya agar
medium and phosphate solubilizing
activity was estimated after 1 to 5 days of
incubation at room temperature. Phosphate
solubilization activity was determined by
the development of the clear zone around
bacterial colony.
Phenotypic characterization of bacterial
isolates
A total of 28 isolates were randomly
selected morphologically from tomato
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Int.J.Curr.Microbiol.App.Sci (2013) 2(10): 406-417
membrane immediately surrounding the
colony (after time of 0.5 to 2 hours) that
the color and size of the colonies varied in
different isolates (depending on the
amount of produced IAA by isolates).
Generally, the tested isolates were group
of IAA hormone production ability as
follows. The halo diameter (HD) and the
colony diameter in strains were measured
and the mean ratio of halo diameter to
colony diameter were calculated for each
isolate and considered as the qualitative
gradation base of IAA production ability.
Production of HCN
All the isolates were screened for the
production of hydrogen cyanide by
performing the method of Lorck (1948).
Briefly, nutrient broth was amended with
4.4 g glycine/l and bacteria were streaked
on modified agar plate. A Whatman filter
paper no. 1 soaked in 2% sodium
carbonate in 0.5% picric acid solution was
placed at the top of the plate. Plates were
sealed with parafilm and incubated at
36±2 °C for 4 days. Development of
orange to red colour indicated HCN
production.
Quantitative assay for Production of
Indole acetic acid
Qualitative assay of IAA Production:
Indole acetic acid (IAA) production was
detected as described by Agrawal et al.,
(2011). Bacterial cultures were grown for
48hours on the nutrient broth at 37±2 °C.
Bacteria were grown overnight in five ml
of nutrient broth supplemented with Ltryptophan to achieve a final concentration
of 0, 50, 100, 200 and 500 g ml-1. After
incubation for 42 h, bacterial growth was
measured spectrophotometrically at 600
nm; the cells were removed from culture
medium by centrifugation at 7,500 rpm for
10 min. 1 ml aliquot of supernatant was
mixed with 4 ml of Salkowski s reagent
(150 ml of concentrated H2SO4, 250 ml of
D.W, 7.5 ml of 0.5 M FeCl3, 6H2O).
Samples were left at 28 20C for 25 min
and absorbance was read at 535 nm. The
concentration of IAA was determined by
referring to a standard curve.
The qualitative assay of IAA produced by
Rhizobacterial strains were conducted on
the basis of Bric s et al. (1991) proposed
method. In this method sterilized Petri
dishes (9-cm diameter) were used. Each
Petri dish was inoculated with toothpicks
and three repeats (3 squares) were
considered for any rhizobacterial isolate.
Then each inoculated Petri dish was
overlaid with an 82-mm-diameter disk of
nitrocellulose membrane. Petri dishes were
inversely incubated into incubator (at
27°C) for 2 to 4 days. Finally when the
diameter of appeared colonies on nutrient
agar media was about 2 mm, IAA
production assay was conducted as
following exposition: The disks of
nitrocellulose
membrane
including
rhizobacterial grown colonies (about 2
mm) were treated with Salkowski reagent
within other Petri dish including a reagent
saturated filter paper (whatman no. 2).
The reaction was allowed to proceed until
adequate colour developed. All the reagent
incubations was conducted at room
temperature. Bacteria producing IAA were
identified by the formation of a
characteristic red halo within the
Evaluation of effective PGPR strains on
seed germination and seedling vigour
Seed germination assay:
The experiment was conducted to assess
the influence of 5 selected efficient
isolates on seed germination and tested for
their plant growth promotion ability by the
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Int.J.Curr.Microbiol.App.Sci (2013) 2(10): 406-417
standard roll towel method (ISTA, 1985)
in growth chamber. Bacteria were grown
in nutrient broth medium on a shaker (150
rpm) for 2 days and centrifuged at 10,000
rpm for 5 minutes. The pellet was mixed
with sterile carboxy methyl cellulose
(CMC) (HiMedia) suspension (1%).
Tomato seeds were surface sterilized with
0.1% mercuric chloride for 5 min, rinsed
with sterilized distilled water (SDW) and
soaked in bacterial suspension (3×108 cfu
ml-1) using 1% carboxymethyl cellulose
(CMC) for 24h and sterile blank nutrient
broth served as control. Then the seeds
were blot dried, placed in wet blotters and
incubated in growth chamber maintained
at 25±20C and 95±3% RH. Each treatment
was replicated four times with 40 seed per
each. The percentage of germination was
recorded at fifth day. Ten seedlings were
taken at random from each replication and
length of root and shoot measured
separately at 6 and 16 days (Abdul Baki
and Anderson, 1973). Plant growth
promotion of tomato seedling was
assessed using Vigour Index (VI).
VI = per cent germination x mean total
length of seedling (root length + shoot
length)
(shape, margin, elevation and surface) and
cell morphology (Gram s reaction, cell
shape and arrangement) and were studied
in detail (table2). Bacteria exhibited wide
morphological
variation.
Maximum
variation in colony shape viz.,irregular,
circular. Maximum of the obtained
bacterial from different soil samples were
Gram Positive Bacilli with Mucoid
texture, entirely edged and creamy white
in appearance as tabulated in table 2.
Plant growth promoting activity of
bacterial isolates
The total isolates (28) obtained in the
present study were mostly gram positive
rods and were subjected to PGPR
properties determining tests (Kloepper et
al., 1980) i.e. siderophore production and
phosphate solublization test. Appearance
of clear zone indicated the positive result
i.e.,
phosphate
solubilization
and
Siderophore production was indicated by
orange halos around the colonies. Out of
28 only 5 isolates showed positive test
namely FAR-IIIb, HBR-II, GAR-III,
HBR-VII and HBS-VIII which were
tabulated. All the isolates were then
subjected to HCN production test
(Rezzonico et al., 2007). Appearance of
reddish brown zone around the inoculated
colony shows positive result. Out of 28
isolates some showed strong results while
other gave moderate and low results.
Isolates which gave negative result had
PGPR property because HCN is a lethal
byproduct which is harmful for the plant
growth promotion. HCN, produced by
many soil microorganisms and it is
postulated to play a role in biological
control of pathogens (Defago et al., 1990).
Production of HCN by certain strains of
fluorescent pseudomonads has been
involved in the suppression of soil borne
pathogens (Siddiqui et al., 2006). The
isolates were also subjected to siderophore
Results and Discussion
A total three rhizospheric and three soil
samples were collected from tomato
growing fields, home and garden in
Dehradun. These samples were plated on
Nutrient agar medium. The total cfu
obtained in these cases were seen to be
maximum in Rhizosphere layer of Garden
soil (3.41 X106 CFU/gram soil) as
compared to Field and Home samples
(table1).
Bacterial morphotypes were selected on
the basis of colour, morphological
characteristics viz., colony morphology
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Int.J.Curr.Microbiol.App.Sci (2013) 2(10): 406-417
Table.1 Functionally active bacterial population associated with tomato crop recorded on
various selective media (CFU g-1 soil)
Sample Type
King s B Media
Nutrient Agar Media
Garden Soil Sample ( I )
1.15X106
2.44 X106
Garden Rhizosphere Sample (I)
Home Soil Sample ( II )
Home Rhizosphere Soil Sample (II)
Field soil sample
Field rhizosphere
3.41 X106
0.49 X106
0.89 X106
0.6 X106
2.38 X106
1.98 X106
1.80 X106
1.40 X106
0.4 X106
0.79 X106
Table.2 Morphotypic characterization of rhizobacteria associated from Tomato
Sample
Name of Grams Reaction
Colour of Margin Elevation
Surface
Form
Isolate
Colony
Morphotypic characterization of rhizobacteria associated from Tomato of Garden Soil Sample
GASIa
Gram Ve, Coccus Off White
Repand Flat
Dry
Irregular
GASIb
Gram +Ve,Shorts Transparent Entire
Convex
Mucoid
Round
thin Rods
GASIIa
Gram Ve, Coccus Creamy
Entire
Raised
Mucoid
Round
White
GASIIb
Gram
+Ve, Creamy
Entire
Umbonat Mucoid
Round
Coccus
White
e
GASIII
Gram +Ve, Short Creamy,Tra Undulat Flat
Dry
Irregular
Rods
nslucent
e
GASIV
Gram
+Ve, Creamy
Erose
Umbonat Glisining
Irregular
Baccillus
White
e
GASVI
Gram +Ve, Short Yellow
Entire
Slightly
Mucoid
Irregular
Rods
Raised
GASVII Gram
+Ve, Off White
Entire
Convex
Mucoid
Round
Coccus
GASIX
Gram Ve, Coccus Orange
Entire
Raised
Mucoid
Irregular
Morphotypic characterization of rhizobacteria associated from Tomato of Garden Rhizo-sphere
Sample
GARI
Gram
+Ve, Off White Cilliate Flat
Dry
Irregular
ShortRods
d
(Streptococci)
GARII
Gram Ve, Cocco White
Cilliate Flat
Dry
Irregular
baccillus
d
GARIII
Gram
+Ve, Off White
Cilliate Flat
Mucoid
Irregular
Baccillus
d
GARIV
Gram
+Ve, White
Entire
Flat
Mucoid
Fusiform
Baccillus
411
Int.J.Curr.Microbiol.App.Sci (2013) 2(10): 406-417
Morphotypic characterization of rhizobacteria associated from Tomato of Home Soil Sample
HBSIII
Gram
+Ve, White
Lobate
Flat
Dry
Irregular
Thin Rods
HBSV
Gram
Ve, Off White
Entire
Flat
Mucoid
Irregular
Spindle Shape
HBSVII
Gram
+Ve, White
Entire
Raised
Mucoid
Circular
Rods
HBSVIII
Gram+Ve,Rods White
Entire
Raised
Dry
Irregular
(Streptococcus)
Morphotypic characterization of rhizobacteria associated from Tomato of Home Rhizos-phere
Sample
HBRI
Gram
+Ve, Creamy
Entire
Umbonat Dry
Irregular
Baccillus
e
HBRII
Gram
+Ve, Off White
Entire
Umbonat Dry
Round
Coccus
e
HBRIII
Gram
+Ve, Off White
Lobate
Convex
Dry
Round
Coccobaccillus
HBRVII
Gram
+Ve, Off White
Entire
Flat
Mucoid
Irregular
Baccillus
Morphotypic characterization of rhizobacteria associated from Tomato of Field Soil Sample
FASIV Gram
+Ve, White
Entire
Raised
Mucoid
Irregular
Strepto Baccillus
FARIa
Gram
Ve, Creamy
Entire
Raised
Mucoid
Round
Coccobaccillus
White
FARIb
Gram
+Ve, Cream
Entire
Raised
Mucoid
Irregular
Coccus
Translucent
FARII
Gram
+Ve, White
Entire
Convex
Mucoid
Round
Coccus (Cluster)
FARIIIa Gram
+Ve, Off White
Entire
Raised
Mucoid
Irregular
Coccus
FARIII Gram
+Ve, Off White
Entire
Raised
Mucoid
Irregular
b
Coccus
FARIV Gram
Ve, Creamy
Entire
Raised
Mucoid
Round
Coccus
Table.3 Plant growth promoting attributes of the Bacterial isolates from the rhizosphere of
tomato plants
Isolate
code
HBS-VIII
FAR-IIIb
HBR-II
GAR-III
HBR-VII
Qualitative assay for IAA
PRODUCTION
+++
+++
++
+++
+++
HCN
production
++
412
Siderophore
production
+
+
+
+
+
P-solublization
activity
+
+
+
+
+
Int.J.Curr.Microbiol.App.Sci (2013) 2(10): 406-417
Fig.1 Production of Indole Acetic Acid ( IAA)
Table.3 Biochemical characterizations of bacterial isolates recovered from
Tomato rhizosphere
Isolate
HBSVIII
HBR-II
FAR-IIIb
GAR-III
HBR-VII
Grams
reaction
+
Endospore
staining
+
Cata
-lase
+
Citrate
utilization
-
Starch
hydrolysis
+
Gelatin
utilization
+
Oxidase
+
Cellulose
utilization
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
-
-
Fig.2 Efficacy of rhizobacterial isolates on seed germination
413
Cellular
morphology
Long
Rods
Bacillus
Long rods
Bacillus
Rods
Motility
+
+
+
+
+
Int.J.Curr.Microbiol.App.Sci (2013) 2(10): 406-417
Fig.3 Efficacy of rhizobacterial isolates on seedling vigour by roll towel method.
Fig.4 Efficacy of rhizobacterial isolates on root length and shoot length by roll towel method
414
Int.J.Curr.Microbiol.App.Sci (2013) 2(10): 406-417
production test. Siderophores are low
molecular
weight,
extracellular
compounds with a high affinity for ferric
iron, that are secreted by microorganisms
to take up iron from the environment (
Sharma and Johri, 2003) and their mode of
action in suppression of disease were
thought to be solely based on competition
for iron with the pathogen (Bakker et al.,
1987;Duijff et al., 1997). On the basis of
above three test screening of isolates were
done.
promotion may be attributed to other
mechanisms such as production of plant
growth promoting hormones in the
rhizosphere and other PGP activities
(Glick, 1995). Production of IAA by
Bacillus is a general characteristic of our
test isolates. Higher level of IAA
production by Pseudomonas was recorded
by other workers (Xie et al 1996).
Production of IAA was indicated by
appearance of pink colour. After
qualitative analysis these isolates were
subjected for quantitative production of
IAA which were tabulated. Isolates
GARIII
showed
maximum
IAA
production and least by isolates FARIIIb
which was obtained manually from
standard graph as shown in fig1. These
isolates (HBS-VIII, FAR-IIIb, HBR-II,
A total five isolates were further selected
and subjected for IAA production test.
(fig1 ). Indole acetic acid (IAA)
production was detected as described by
Brick et al., (1991).The ability of bacteria
to produce IAA in the rhizosphere depends
on the availability of precursors and
uptake of microbial IAA by plant. Growth
GAR-III and HBR-VII) were then
subjected to biochemical characterization
for the determination of probable
microorganism which were tabulated.
Bacillus species were estimated by
morphologies
and
physiology
characteristics based techniques table3.
HBRVII showed significantly increased
seed germination (97.5%) and also shoot
and root length as well as enhanced vigour
index of 115.50, 714.35 after 6 and 16
days respectively.
Acknowledgement
These isolates were then assessed to know
their influence on the seed germination by
measuring the shoot and root length. The
growth promoting activity of 5 isolates of
PGPR was tested for seed germination and
seedling vigour by using tomato seeds in
roll towel methods. The data on seed
germination and seedling vigour of tomato
as influenced by seed bacterization with
different rhizobacterial isolates by roll
towel method are given in Fig 2,3,4 In
general, the seed germination ranged from
90 per cent (uninoculated control) to 97.5
percent (HBRVII). All the strains,HBSVIII, FAR-IIIb, HBR-II, GAR-III and
HBR-VII significantly improved seed
germination when compared to the
uninoculated control (UIC). The isolate
The author wish to thank to chairman Mr
Harish Arora and Vice chairperson Mrs.
Rani arora of SIPAS, Dehradun. We
acknowledge the sincere thanks to Dean
life science Prof V D Sharma and
Principal SIPAS Dr. Sandhya Dogra for
providing us research facilities.
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