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Int.J.Curr.Microbiol.App.Sci
(201
4)
3(5):
432
-
447
432
Original Research Article
Evaluating the diversity and phylogeny of plant growth promoting bacteria
associated with wheat
(
Triticum aestivum
)
growing in central zone of India
Priyanka Verma
1
,2
, Ajar Nath Yadav
1,
Sufia
Khannam
Kazy
2,
Anil
Kumar
Saxena
1
and Archna Suman
1*
1
Division of Microbiology, Indian Agricultural Research Institute, New Delhi
- 110012, India
2
Department of Biotechnology, National Institute of Technology, Durgapur
-713209, India
*
Corresponding author
A B S T R A C T
Introduction
Wheat (Triticum durum L.
)
is one of the
most important cereals world-wide and it
is grown in different environments.
Drought is one of the major constraints
which hamper wheat produc
tion in India.
The central zone of India (Madhaya
Pradesh, Kota region of Rajasthan and
Jhansi region of Uttar Pradesh) were
characterised for water stress ecosystem.
Plant growth promoting bacteria (PGPB)
ISSN: 2319
-7706
Volume
3
Number
5
(201
4
) pp.
432
-
447
http://
www.ijcmas.com
Keyw or ds
Epiphytic;
Endophytic;
Rhizospheric;
PGPB;
Drought
stress;
Biocontrol
The diversity of plant growth promoting bacteria was investigated from wheat
growing in different sites in central zone of India. Epiphytic, endophytic and
rhizospheric bacteria were isolated using different growth medium. Bacterial
diversity was analysed through amplified ribosomal DNA restriction analysis
(ARDRA) using three restriction enzymes
Alu
I,
Hae
III, and Msp I which led to
the grouping of 348 isolates into 24-29 clusters at >75% similarity index.
16S
rRNA gene based phylogenetic analysis, revealed that 134 strains belonged to three
phyla namely
actinobacteria,
firmicutes
and proteobacteria with 38 distinct sp
ecies
of 17 genera.
Bacillus
and
Pseudomonas
were dominant in rhizosphere while
Methylobacterium
were in phyllosphere. Endophytic niche specific bacteria were
identified as
Delftia
and
Micrococcus
. Sampling of different sites showed variation
in diversity
indices.
In vitro plant growth promoting activities of bacteria exposed
more than three beneficial traits which may act independently or concurrently.
Phosphate solubilization and siderophores production are the predominant traits
exhibited by these microbes. The many species of genera
Bacillus,
Exiguobacterium, Micrococcus, Pseudomonas
and
Psychrobacter
showed
antagonistic properties against fungal pathogens Fusarium graminerum
,
Rhizoctonia solani and Macrophomina phaseoli. These promising isolates showing
a range of useful plant growth promoting attributes insist to be explored for
agricultural applications.
Int.J.Curr.Microbiol.App.Sci
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are free-living soil, rhizospheric, e
piphytic
and endophytic bacteria that can either
directly or indirectly have an impact on
plant growth (Glick et al. 1999; Verma et
al. 2013). PGPB stimulate plant growth in
multiple ways
viz
N2 fixation, synthesize
phytohormones (auxin and cytokinin),
pro
duction of siderophores and suppress
pathogenic organisms. PGPB has been
reported not only to improve plant growth
but also to suppress the plant pathogens, of
which Pseudomonas
and Bacillus
were
well characterised.
Pink
-
pigmented
facultative methylotrophs synthesize a
variety of metabolites useful for the
plants
including phytohormones (Ivanova et al.
2001; Verma et al. 2013) that promote
plant growth and yield. PGPB are used as
biological control agents to reduce the
development of plant diseases caused by
plant pathogenic fungi, bacteria, viruses
and nematodes.
In the last decade, a number of PGPB
associated with wheat and different cereals
crops have been identified including
Acinetobacter, Arthrobacter, Azospirillum,
Azotobacter, Bacillus, Burkholderia,
Citricoccus, Kocuria, Lysinibacillus,
Methylobacterium,
Paenibacillus,
Providencia, Pseudomonas
and
Serratia
(Coombs and Franco 2003; Conn and
Franco 2004; Streptomyces 2005; Jha and
Kumar 2009; Wellner et al. 2011; Meena
et al. 2012; Verma et al. 2013)
. The
phyllosphere
is common niche for
synergism between bacteria and plant.
Many bacteria such as Pseudomonas and
Methylobacterium
have been reported in
the wheat phyllosphere (Wellner et al.
2011; Meena et al. 2012; Verma et al.
2013)
. Rhizospheric bacteria have the
ability to attach to the root surfaces
(rhizoplane) allowing these to derive
maximum benefit from root exudates.
Endophytic bacteria live in plant tissues
without causing substantive harm to the
host.
Bacterial endophytes such as
Achromobacter
, Microbiospora,
Micrococcus,
Micromomospora,
Pantoea
,
Planomonospora, Pseudomonas,
Stenotrophomonoas, S
treptomyces
and
Thermomonospora
have been reported
from wheat (Zinniel et al. 2002; Coombs
and Franco 2003; Verma et al. 2013).
A
number of bacterial species associated
with the wheat rhizosphere were recovered
belonging to genera
Azospirillum,
Arthrobacter,
Acinetobacter,
Bacillus,
Burkholderia,
Enterobacter,
Erwinia,
Flavobacterium,
Methylobacterium,
Pseudomonas,
Rhizobium
and Serratia
(Xie et al. 1996; Lavania et al. 2006;
Chaiharn and Lumyong 2011; Meena et al.
2012; Verma et al. 2013)
.
The present study attempted to elucidate
the bacterial diversity associated with
wheat growing in central zone of India,
employing different growth media.
ARDRA analysis was done for
phylogenetic clustering of the moderately
drought tolerant isolates. Sequencing the
16S rRNA gene of representative strains
was undertaken for identification.
Representative strain from each cluster
was screen in vitro for plant growth
prom
otion in drought stress condition.
PGPB inoculants are inexpensive, simple
to use and have no unpleasant effects to
land. The use of PGPB may prove useful
in developing strategies to facilitate wheat
and other cereals crops growth in drought
area.
Materi
als and Methods
Samples collection
Wheat plants of two
var.
IWP5007 and
HD2987
with rhizospheric soil were
collected from five different sites in
Int.J.Curr.Microbiol.App.Sci
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central zone of India, which included
Gwalior, Sagar and Indore region of
Madhya Pradesh, Jhansi region of
Uttar
Pradesh and Kota region of Rajasthan
(Table 1). A total forty samples, eight
from each site were collected in sterile
polythene bags labelled transported on ice
and processes immediately.
Physico
-
chemical properties of samples
The pH and conductivity of the soil
samples were recorded on sampling site.
Soil samples analyzed for soil organic
carbon, total nitrogen (%), soil organic
matter, exchangeable cations and available
phosphorus was determined as described
earlier
Verma et al. (2013). Soil anal
ysis
was done at Division of Soil Sciences,
Indian Agricultural Research Institute,
New Delhi, India.
Enumeration of wheat associated
bacteria
The culturable bacteria from soil and
rhizosphere soil were isolated through
enrichment using the standard seri
al
dilution plating technique employing nine
different growth
media as described earlier
(Verma et al. 2013)
.
Endophytic and
epiphytic bacteria were isolated using
methods described by Conn and Franco
(2004)
and Holland and Polacco (1994)
respectively
. The plates were incubated at
30 °C and the population was counted
after 3-7 days. Colonies that appeared
were purified by repeated re-streaking to
obtain pure colonies using respective
medium plates. The pure cultures were
maintained at 4 °C as slant and glycerol
stock (25 %) at -80 °C for further use. All
the isolates were screened in
triplicates
for
tolerance to pH and drought [Low water
potential on polyethylene glycol (PEG-
8000)
- infused plates] as described earlier
Yadav et al. (2014).
PCR amplification of 16S rDNA and
amplified rDNA restriction analysis
(ARDRA)
Genomic DNA was extracted by the
method as earlier described by Kumar et
al.
(2013).
The amount of DNA extracted
was electrophoresed on 0.8% agarose gel.
Amplification of 16S rDNA was done by
us
ing the universal primers pA (5 -
AGAGTTTGATCCTGGCTCAG
-3 ) and
pH (5 -
AAGGAGGTGATCCAGCCGCA
-
3 ). The amplification was carried out in a
100 l volume and
amplification
conditions were used as described earlier
(Pandey et al. 2013). The PCR amplified
16S rDNA were purified by QIA quick
PCR product purification kit (Qiagen). 100
ng
purified PCR products were digested
separately with three restriction
endonucleases
Alu
I,
Hae
III and
Msp
I
(GeNei) in 25 l reaction volumes, using
the manufacturer s recommended b
uffer
and temperature. The clustering analysis
was undertaken using the software,
NTSYS
-2.02e package (Numerical
taxonomy analysis program package,
Exeter software, USA). Similarity among
the isolates was calculated by Jaccard s
and dendrogram was constructed using the
UPGMA method
(Nei and Li 1979)
.
16S rDNA Sequencing and phylogenetic
analysis
PCR amplified 16S rRNA genes were
purified and sequenced using both pA and
pH primers for forward and reverse
reactions respectively. Sequencing
employed a dideox
y cycle with fluorescent
terminators and was run in a 3130xl
Applied Biosystems ABI prism automated
DNA sequencer (Applied Biosystems,
Foster City, CA) at SCI Genome Chennai,
India.
16S rRNA gene sequences were
analysed using codon code aligner v.4.0.4.
Int.J.Curr.Microbiol.App.Sci
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Th
e 16S rRNA gene sequences were
aligned to those of closely related bacterial
species available at GenBank database
using BLASTn program. Bacterial isolates
were identified based on percentage of
sequence similarity ( 97%) with that of a
prototype strain sequence in the GenBank.
The phylogenetic tree was constructed on
the aligned datasets using the neighbour-
joining
method
(Saitou and Nei 1987)
implemented in the program MEGA 4.0.2
(Tamura et al. 2007). Bootstrap analysis
was performed as described by
Felse
nstein
(1981)
on 1000 random samples taken
from the multiple alignments. The partial
16S rRNA gene sequences were submitted
to NCBI GenBank and accession numbers
were assigned
from KF054878
-
KF054913
and
KF572999
-
KF573001.
In vitro screening of isolates for PGP
traits
Representative isolates from each cluster
were screened for PGP attributes
initially
as
qualitative estimation
for
in vitro
production of ammonia (Cappucino 1992),
siderophore
(Schwyn and Neilands 1987),
HCN
(Bakker and Schippers 1987),
gibb
erllic acid (Brown and Burlingham
1968)
and indole-3-acetic acid (Bric et al.
1991)
. The strains were screened for
solubilization of phosphorus
(Pikovskaya
1948),
potassium
(Hu et al. 2006) and zinc
(Saravanan et al. 2004). The ability to fix
nitrogen was evaluated using semi-
solid
nitrogen
-free NFb medium (Dobereiner et
al. 1996)
.
The bacterial strains were
screened for their ability to utilize the 1-
aminocyclopropane
-1-carboxylate (ACC)
as sole nitrogen source, a trait that is
consequence of the activity of the enzyme
ACC deaminase (Jacobson et al. 1994).
In
vitro
antagonistic activity of bacterial
isolates was evaluated against three fungal
pathogens
Fusarium graminerum,
Rhizoctonia solani and
Macrophomina
phaseoli
according to the method
described by
Ver
ma et al. (2013)
.
Statistical Analysis
In order to compare the bacterial diversity
among five different sites, the 16S rRNA
gene sequences of the isolates showing
97 % sequence similarity were grouped
into the same OTU (phylotype). The
software Shannon Wiener Diversity
Index/Shannon Entropy Calculator
(http://www.changbioscience.com/genetic
s/shannon.html) and
Rarefaction
Calculator (
http://www2.biology
.ualberta.
ca/ jbrzusto/rarefact.php) were used to
calculate Shannon index (H), Evenness (J)
and the Simpson s index (D). Principal
coordinate analysis (PCA) was used to
determine the statistical correlation
between population diversity of five sites
survey (
Rico et al. 2004)
.
Results and Discussion
Enumeration and characterization of
wheat associated bacteria
A total of 348 bacteria were isolated from
five different sites in central zone of India
(Table 2). Significant variations were
observed among the culturable bacterial
population (CFU) of each sample on
different
growth media. The Population of
bacteria varied from 2.1 ×106 to 7.8×106,
1.2×10
6 to 8.8×106, 1.0×106 to 1.8×10
6
for
isolation sources of phyllosphere,
rhizosphere and endophytic respectively
(Table 2).
The pure colonies obtained from
each sample on different media were
isolated based on colony morphology and
cultural characteristics. The representative
strains were screened for tolerance to
range of pH and PEG mediated water
deficit (drought). Among the 38 isolates,
22 were able to grow on PEG-8000
infus
ed plates with water potential of
-0.25
Int.J.Curr.Microbiol.App.Sci
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Mpa and 11 isolates tolerates upto -0.5
Mpa (
Fig. 5).
Physico
-
chemical properties of samples
Physical and chemical characteristics of
the soil varied considerably amongst the
different soil samples
(Table1).
Availa
ble
nitrogen content was highest in
Gwalior
sample and it
ranges from 149
-177
kg ha
-1.
Organic carbon content was significantly
higher, achieving of 4.9 % organic carbon
in
Indore followed by 4.3 % in Gwalior
samples
(Table 1).
PCR amplification of 16S rDNA and
ARDRA
PCR amplification of 16S rDNA followed
by ARDRA with three restriction
endonucleases was carried out to look for
the species variation among the
morphotypes selected. The 16S rDNA
amplicons were digested with restriction
enzymes, which generated profiles having
3 to 7 fragments ranging in size from 100
to 860 base pairs.
ARDRA results revealed
th
at among the restriction endonucleases,
Alu
I was more discriminatory as
compared to
Msp
I and
Hae
III. A
combined dendrogram was constructed for
each
sampling site to determine the
percent similarity among the isolates. At a
level of 75 % similarity, the isolates were
grouped into clusters; and the number of
clusters ranged from 24 (for JCZ) to 29
(for ICZ). The total number of clusters
was 134, summed up for all the sites
(Table 2).
16S rRNA gene sequencing and
phylogenetic analysis
16S rRNA gene sequencing and
phylogenetic analysis of a representative
isolate from each cluster revealed that all
the isolates showed > 97 to 100 %
similarity with the sequences within the
GenBank (Table 3). One sequence from
each group was selected as a
representative operational taxonomic unit
(OTU) and all the isolates were
classified
into 38 OTUs using a >
97 % sequence
similarity cut-off value. The phylogenetic
tree
of 38 identified bacteria was
constructed to determine their affiliations
(Fig.1).
Analysis of the 16S rRNA gene
sequences revealed that 134 strains
belonged to 3 phyla namely
actinobacteria
(18 %), firmicutes (38 %) and
proteobacteria (43%) with 38 distin
ct
species of 17 genera (Table 3; Fig. 2a, b).
Three major clusters were formed in
which proteobacteria were most pre-
dominant phylum followed by firmicutes.
Out of the 38 OTUs, 17 strains belonged
to phylum firmicutes were grouped into
three families of bacilli namely
Bacillaceae (11 strains Bacillus subtilis,
Bacillus alcalophilus, Bacillus aquimaris,
Bacillus aryabhattai, Bacillus barbaricus,
Bacillus cereus, Bacillus megaterium,
Bacillus pumilus, Bacillus tequilensis,
Bacillus thuringiensis
and
Lysini
bacillus
xylanilyticus
); Bacillales incertae sedis (1
strain
Exiguobacterium acetylicum)
and
Paenibacillaceae
(5 strains Paenibacillus
amylolyticus, Paenibacillus
dendritiformis, Paenibacillus durus,
Paenibacillus
sp.
and
Paenibacillus
tundrae
). Second cluster of phylum
actinobacteria consist of five
strains,
Arthrobacter humicola, Corynebacterium
callunae, Kocuria
sp.,
Micrococcus luteus
and Micrococcus
sp. (Fig. 1). Phylum
proteobacteria consist three grouped of
alpha proteobacteria (3 strain
Methylobact
erium extorquens,
Methylobacterium mesophilicum
and
Methylobacterium radiotolerans), beta
proteobacteria (2
strains
Duganella
Int.J.Curr.Microbiol.App.Sci
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437
violaceusniger
and
Delftia
sp.) and gamma
proteobacteria (11 strains Acinetobacter
sp.
, Pantoea ananatis, Pseudomonas
fuscovaginae
, Pseudomonas lini,
Pseudomonas monteilii, Pseudomonas
stutzeri, Pseudomonas thivervalensis
,
Psychrobacter fozii, Serratia marcescens,
Stenotrophomonas maltophilia
and
Stenotrophomonas sp.) (Fig. 1).
Overall
Micrococcus
from actinobacteria, Bacillus
from f
irmicutes,
and
Pseudomonas
from
proteobacteria
were
the most frequently
recovered genera (Table 3).
Statistical analysis
The 134 isolates from the five sampling
sites based on similarity index of > 97 %
at the 16S rRNA gene sequences could be
categorised
into 24-29 clusters (Table 4).
Shannon s diversity index was recorded
highest (H´=3.3) for Kota and
lowest
(H´=3.12) value for Jhansi using 16S
rRNA sequences and ARDRA data. The
highest species richness was recorded in
Indore
(Table 4). The individual
ra
refaction curves for all the five sites
indicated that the bacterial populations
were the least diverse in Jhansi and most
diverse in Indore (Fig. 3).
Principal
coordinate analysis was used to investigate
relationships between bacterial diversity
(Shannon s diversity index). The first two
dimensions of PCA (PCA1 and PCA2)
explained 67.29 % of the total variation,
with component 1 accounting for 48.42 %
and component 2 for 18.87 % of the
variance (Fig. 4).
Plant growth promoting (PGP)
attributes
Out of 38 representatives, 29, 10 and 21
strains were positive for solubilisation of
phosphorus, zinc and potassium
respectively (Table 3). Production of
siderophores, IAA, gibberellic acid and
ammonia were positive for 21, 17, 12 and
21 strains respectively (Table 3). Nine
strains showed nitrogen fixation confirmed
by acetylene reduction assay. ACC
deaminase activity was shown by 9 strains.
Isolate IARI
-
IIWP
-20 solubilised highest
amount of phosphorus (326±1.5 µg
mg
-
1
day
-1) followed by
IARI
-
IHD
-5 (
126±1.2
µg
mg
-1d
ay
-1
).
Isolate
IARI
-
IHD
-3 showed
highest IAA production
(
280.4±0.5
µg mg
-
1
protein day-1
)
followed IARI
-
IIWP
-2
(
102.8±05
µg mg-
1
protein day-1). Highest
solubilization of potassium by isolates
IARI-
IIWP
-12 (3.8±0.8 mm). Isolate
IARI-
IHD
-4 show highest zinc
solubilization (9.8±1.5 mm). Of 38 stains,
11 strains were anatagonastic against
Fusarium graminerum, Rhizoctonia solani
and
Aspergillus fumigatus
(Table 3).
Bacteria associated with wheat have been
frequently isolated and identified as
endophytic and rhizobacteria but this
paper provides the diversity of bacteria
present in endophytic, epiphytic as well as
rhizospheric. Representative strains were
screened for eleven different plant growth
promoting attributes including
solubilization of phosphorus, pota
ssium
and zinc; production of ammonia,
gibberellic acid, HCN, IAA, siderophores;
Nitrogen fixation and ACC deaminase
activity.
Invitro
antagonistic activity was
done against three pathogenic fungus
Fusarium graminerum, Rhizoctonia solani
and
Aspergillus fu
migatus
.
In present study we have isolated wheat
associated bacteria (Epiphytic, endophytic
and rhizospheric) form five locations in
central zone (One of the wheat agro-
ecological zones) in India. From the
phyllosphere a total of 89 bacteria isolated,
bel
ong to different genera of
Arthrobacter,
Bacillus, Corynebacterium,
Methylobacterium, Paenibacillus,
Pseudomonas
and
Psychrobacter.
Pink
-
Int.J.Curr.Microbiol.App.Sci
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pigmented facultative methylotrophs
(PPFMs)
Methylobacterium extorquens,
Methylobacterium mesophilicum and
Methylobacte
rium radiotolerans are a
physiologically and taxonomically diverse
group of bacteria with prominent plant
growth promoting attributes (Verma et al.
2013)
. The genus
Methylobacterium
is
among the commonly recorded leaf epiphytes
and represents abundant and stable members
of the phyllosphere community of a wide
range of crop plants (Holland and Polacco
1994; Meena et al. 2012; Verma et al. 2013)
.
A total of 222 rhizospheric bacteria were
isolated, belonged to twelve genera namely
Acinetobacter, Bacillus, D
uganella,
Exiguobacterium, Kocuria, Lysinibacillus,
Micrococcus, Paenibacillus, Pantoea,
Pseudomonas, Serratia
and
Stenotrophomonas
(Table 3). Thirty seven
endophytic bacteria were isolated and
identified belonging to genera of
Delftia,
Micrococcus, Pseudo
monas
and
Stenotrophomonas
(Table 3).
Among plant growth promoting activities, P-
solubilization were highest (17 %) when
compared to zinc solubilization (13 %),
ammonia, IAA and siderophore production
(12 %), Biocontrol and GA ( 7%), K-
solubilization (6%), Nitrogen fixation, ACC
deaminase (5 %) and HCN production (4%).
Among PGPR, members of
Bacillus
and
Bacillus
derived genera (BBDG) are
ubiquitous in nature that included both free-
living PGPR and pathogenic species. PGPR
belonging to BBDG have been reported to
enhance the growth of several plants such as
wheat, tomato, sugar beet, sorghum and
peanut. A next to BBDG, another group of
PGPR belonged to the genus
Pseudomonas
(Yadav et al. 2013; Verma et al. 2013)
.
Previously it is reported that
Pseudomonas
PGPR are highly resistant to various
environmental stresses (Paul and Nair 2008)
.
Production of ACC deaminase by
Pseudomonas fluorescence increases the
resistance of plants to salt stress (Sandhya et
al. 2010)
.
Table.1
Sampling sites and physico
-
chemical
properties of soil
Sampling sites
pH
EC
(mS/cm)
%OC
Avail. N
(kg ha
-1)
Avail. P
(kg ha
-1)
Avail. K
(kg ha
-1)
Zinc
(mg kg
-1)
Exch. Na
(mg kg
-1)
Gwalior GCZ)
7.8
-
8.3
46.8
4.3
177
13.7
1286
1.50
51.83
Sagar (SCZ)
7.2
-
8.2
49.2
4.0
167
11.2
1119
1.36
45.33
Indore (ICZ)
7.2
-
8.5
47.9
4.9
163
12.5
1245
1.39
49.23
Jhansi (JCZ)
7.1
-
7.9
41.3
3.8
156
11.1
1086
1.12
39.85
Kota (KCZ)
7.4
-
8.9
40.2
3.4
149
10.3
1036
1.02
39.28
Table.2
Total
viable count of bacteria associated with wheat growing in different site f
rom central
zone of India
Total viable count (
CFU g
-1
soil
× 10
6
) on different media*
Epiphytic
Rhizospheric
Endophytic
Sampling
sites
NA
AMS
TSA
NA
T3A
KB
JA
SEA
TSA
LB
MDM
M#
GCZ
5.3
3.4
7.8
8.8
2.9
4.7
2.2
4.9
4.5
1.2
1.3
60
SCZ
5.2
3.4
7
.2
8.4
2.6
4.2
2.7
4.1
4.0
1.5
1.4
59
ICZ
5.8
3.9
7.6
7.9
2.8
4.4
2.5
4.4
4.7
1.8
1.6
80
JCZ
4.2
2.3
6.5
6.4
2.1
3.7
1.6
3.2
3.6
1.0
1.2
53
KCZ
4.8
2.1
6.2
6.3
1.7
3.2
1.2
3.7
3.1
1.0
1.1
66
*Ammonium minerals salt (AMS); Jensen s agar (JA); King s B agar (KB); Luria bertani agar (LB);
Modified
Dobereiner medium (MDM); Nutrient agar (NA); Soil extract agar (SEA); T3 agar (T3A); Tryptic soy agar
(TSA)
M#-
Total morphotypes
.
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Fig.1
Unrooted phylogenetic tree based on the 16S ribosomal DNA sequences of bacteria
isolated from wheat growing in central zone of India. The trees were constructed
using Neighbor joining with algorithm using MEGA4 software (Tamura et al. 2007).
One thousand bootstrap replicates were performed. Bootstrap values are indicated on
the branches.
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447
440
Table.3 Identification and functional attributes of the bacterial isolates associated with
wheat growing in central
zone
of India
Solubilization
Strain number
Nearest phylogenetic relative
Similarity
(%)
Bacteria type
Phospho
rus
*
Potassium
$
Zinc
$
IARI
-
IIWP
-
20
Micrococcus
sp.
99
E
326±1.5
- -
IARI
-
IIWP
-
24
Corynebacterium callunae
100
P - -
3.5±0.5
IARI
-
IIWP
-
42
Arthrobacter humicola
100
P
61.9±0.2
-
2.4±0.1
IARI
-
IHD
-5
Micrococcus luteus
99
R
126±1.2
- -
IARI
-
IHD
-9
Kocur
ia
sp.
100
R - -
5.3±1.2
IARI
-
IIWP
-2
Bacillus subtilis
99
R
24.8±0.8
-
7.0±1.0
IARI
-
IIWP
-4
Paenibacillus dendritiformis
100
R
58.9±0.7
3.3±1.2
9.8±2.1
IARI
-
IIWP
-7
Paenibacillus tundrae
99
R - -
2.2±0.5
IARI
-
IIWP
-9
Bacillus megaterium
99
R
45.7±1.1
2.2±0.5
4.3±0.2
IARI
-
IIWP
-
14
Bacillus cereus
99
R - - -
IARI
-
IIWP
-
15
Bacillus tequilensis
99
R - - -
IARI
-
IIWP
-
25
Lysinibacillus xylanilyticus
100
R - -
2.8±0.8
IARI
-
IIWP
-
38
Bacillus thuringiensis
99
R
63.2±1.4
-
3.1±0.1
IARI
-
IIWP
-
40
Paenibacillu
s durus
100
R
32.3±1.4
- -
IARI
-
IHD
-
10
Bacillus barbaricus
100
R - -
2.3±0.8
IARI
-
IHD
-
15
Paenibacillus
sp.
99
R
56.2±0.6
- -
IARI
-
IHD
-
17
Bacillus aquimaris
99
R
326±1.5
- -
IARI
-
IHD
-
21
Exiguobacterium acetylicum
100
R
9.9±1.0
-
6.1±1.2
IARI
-
IHD
-22
Bacillus alcalophilus
100
R
24.9±0.8
-
2.0±0.5
IARI
-
IHD
-
23
Bacillus pumilus
100
R
21.5±1.0
- -
IARI
-
IHD
-
24
Paenibacillus amylolyticus
100
P
24.4±1.0
2.8±1.2
IARI
-
IHD
-
34
Bacillus aryabhattai
100
P
45.6±1.0
- -
IARI
-
IIWP
-
43
Methylobacterium extorq
uens
99
P
23.6 ± 1.0
- -
IARI
-
IIWP
-
45
Methylobacterium mesophilicum
100
P
12.6 ± 1.5
- -
IARI
-
IHD
-
35
Methylobacterium radiotolerans
98
P
14.6 ± 1.2
- -
IARI
-
IIWP
-
23
Duganella violaceusniger
99
R
58.9±0.7
3.3±1.2
5.3±2.1
IARI
-
IIWP
-
31
Delftia
sp.
100
E
32.2±1.4
-
6.3±0.6
IARI
-
IHD
-3
Pseudomonas thivervalensis
100
R
73.5±0.6
2.0±0.5
3.8±0.5
IARI
-
IHD
-4
Pseudomonas stutzeri
99
R - -
7.8±1.5
IARI
-
IHD
-
30
Pantoea ananatis
100
R - -
2.1±0.1
IARI
-
IIWP
-1
Acinetobacter
sp.
99
R
21.6±1.0
- -
IARI
-
IIWP
-
12
P
sychrobacter fozii
99
P
20.83±1
3.8±0.8
5.8±1.5
IARI
-
IIWP
-
18
Stenotrophomonas maltophilia
99
E
55.7±0.5
2.8±1.2
-
IARI
-
IIWP
-
27
Pseudomonas monteilii
100
E
40.5±0.4
3.3±0.5
6.3±1.5
IARI
-
IIWP
-
29
Pseudomonas fuscovaginae
99
P
29.2±0.8
-
7.7±0.6
IARI
-
II
WP
-
32
Serratia marcescens
99
R
46.6±0.9
- -
IARI
-
IIWP
-
33
Pseudomonas lini
99
R
55.9±0.6
3.3±0.5
8.3±0.6
IARI
-
IIWP
-
34
Stenotrophomonas
sp.
99
R
23.7±0.5
2.2±0.5
-
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-
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441
Table 3
(Continued)
Production
Other activities
Strain number
IAA*
Siderophore
$
GA
HCN
NH
3
ACC
N
2
Fixation
Bio
control
IARI
-
IIWP
-
20
- - + - - - - +
IARI
-
IIWP
-
24
-
2.40±0.5
- - - - - -
IARI
-
IIWP
-
42
27.8±1.2
1.0±0.1
- - + - + -
IARI
-
IHD
-5 - - + - - - - +
IARI
-
IHD
-9
32.6±2.6
- - - + + -
IARI
-
IIWP
-2
102.8±0.5
5.3±0.5
+ - - + - -
IARI
-
IIWP
-4
45.2±1.1
4.7±0.5
+ - + - - -
IARI
-
IIWP
-7 - - - - + - - -
IARI
-
IIWP
-9
16.6±1.0
3.5±0.2
- - + - - -
IARI
-
IIWP
-
14
-
2.6±0.1
- - - - - -
IARI
-
IIWP
-
15
-
22.5±0.5
+ - + +
IARI
-
IIWP
-
25
- - - - + + - -
IARI
-
IIWP
-
38
-
2.5±0.1
- + + - - -
IARI
-I
IWP
-
40
- - - - + + + +
IARI
-
IHD
-
10
35.2±1.6
- - - - - - +
IARI
-
IHD
-
15
30.8±1.1
4.8±1.2
- - + - - +
IARI
-
IHD
-
17
- - + - - - - +
IARI
-
IHD
-
21
- - - - - - - +
IARI
-
IHD
-
22
- - - - - - +
IARI
-
IHD
-
23
- - - + - - - -
IARI
-
IHD
-
24
- - - - - - - -
IARI
-
IIWP
-
43
16.2±1.1
3.5±0.2
- - + - - -
IARI
-
IIWP
-
45
12.1±1.2
4.5±0.1
- + + - - -
IARI
-
IHD
-
35
11.6±1.3
2.5±1.2
- - + - - -
IARI
-
IHD
-
34
15.6±0.7
2.5±0.1
+ + - - + -
IARI
-
IIWP
-
23
45.17±1.1
4.7±0.5
+ - + - - -
IARI
-
IIWP
-
31
21.5±1.1
9.7±0.9
- + - - + -
IARI
-
IHD
-3
280.4±0.5
2.2±0.5
+ - + + + -
IARI
-
IHD
-4 -
6.8±0.8
+ - + - - -
IARI
-
IHD
-
30
-
4.6±0.1
- - - - - -
IARI
-
IIWP
-1
15.2 ±0.4
- - + - - + -
IARI
-
IIWP
-
12
65.9±1.0
2.8±1.5
+ - + + +
IARI
-
IIWP
-
18
66.1±0.7
2.4±0.1
- - + - -
IARI
-
IIWP
-
27
35.7±0.7
6.0±0.8
- - + + + -
IARI
-
IIWP
-
29
28.5±1.1
7.0±0.8
- - + - + +
IARI
-
IIWP
-
32
- - + - - - - -
IARI
-
IIWP
-
33
-
4.7±1.2
- - - + - -
IARI
-
IIWP
-
34
36.1±0.7
- - - + - + -
P-
Phyllospheric; E
-
Endophytic; R
-
Rhizospheric; K
-
Potassium; IAA
-
Indole 3
-
acetic acid;
GA
-
Gibberell
ic acid ACC
-1-
aminocyclopropane
-1-
carboxylate; *Numerical values are mean
± SD of three independent observations; Phosphate (µg mg
-
1day
-
1); IAA (µg mg
-
1 protein
day
-
1); # Radius of halo zone in mm;
-, negative for the attributes; +, positive for the
attrib
utes
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442
Table
.4 Diversity indices for the isolates associated with wheat from five sites in
central
zone
of India
GCZ
SCZ
ICZ
JCZ
KCZ
No of isolates
68
64
87
59
70
Species richness
27
26
29
24
28
Evenness (J´)
0.95
0.94
0.94
0.94
0.97
Shannon (H)
3.25
3.20
3.31
3.12
3.30
Simpson s (D)
0.96
0.95
0.96
0.95
0.96
Chao
-1
27
26.27
29
24
28
Fig. 2
Abundance
of different bacteria; a Distribution of phylum and group in the samples
surveyed;
b Distribution of total bacteria isolated from five different site of central zone of
India
Int.J.Curr.Microbiol.App.Sci
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443
Fig. 3
Rarefaction curves of observed OTUs in wheat associated bacterial isolates from five
sites of
central zone of India using 16S rRNA sequencing analysis
Fig. 4 Principal coordinate analysis (PCA) of the diversity indices (H) of the16S rRNA PCR-
ARDRA profiles of the five sites in relation to 16S rRNA gene sequences, Component 1 and
component 2 accounted for 48.42 % and for 18.87 % of the total variation, respectively.
Int.J.Curr.Microbiol.App.Sci
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444
Fig. 5
Distribution of bacterial isolates b
ased on their
degree of
tolerance and belonging to
five
different sits in central zone of
India,
with respect to water stress
and pH.
Microbes associated with plants can be
harmful and beneficial. PGPB promote
growth directly by nitrogen fixation,
solubi
lization phosphorus and potassium,
production of siderophores, ammonia,
HCN and production of plant growth
hormones (cytokinin, auxin and gibberellic
acid)
(Tilak et al. 2005). Many bacteria
support plant growth indirectly by
improving growth restricting c
onditions
via production of antagonistic substances.
A number of bacterial species associated
with the plant belonging to genera
Stenotrophomonas, Serratia,
Psychrobacter, Pseudomonas, Pantoea,
Paenibacillus, Micrococcus,
Lysinibacillus, Kocuria, Exiguobac
terium,
Duganella, Delftia, Corynebacterium,
Bacillus, Arthrobacter and Acinetobacter
are able to exert a beneficial effect on
plant growth.
Phosphate (P) and potassium (K) are the
major essential macronutrients for
biological growth and development. The
most efficient phosphate solubilizing
bacteria (PSB) belong to genera
Bacillus
and
Pseudomonas.
There are considerable
populations of P- or K-
solubilizing
bacteria in soil and in plant rhizospheres.
P-solubilizing bacteria (PSB) have ability
to solubilize inorganic phosphate
compounds
(Goldstein 1995). K-
solubilizing bacteria (KSB) were found to
resolve potassium, silicon and aluminium
from insoluble minerals (Hu et al. 2006)
.
Zinc is a nutrient at low concentration but
toxic at higher concentration. The
so
lubilization of zinc might limit the
growth of the bacteria at higher level. Zinc
solubilization by bacteria has an immense
importance in zinc nutrition to plants.
Indole
-3-acetic acid (IAA) is
phytohormones, a type of best
characterized auxin, which is essential for
the growth and development of plants. The
capacity to synthesize IAA is widespread
among soil- and plant associated bacteria.
It has been estimated that 80% of bacteria
isolated from the rhizosphere can produce
the plant growth regulator IAA
(P
atten and
Glick 2002)
.
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In conclusion, utility of bacterial strains in
the context of semi arid agro ecosystems is
immense considering the unique crop
growing situations and the climatic
conditions of the drought agricultural
systems. Such systems require situation-
specific microbial inoculants that
withstand extremities of alkali and retain
their functional traits for plant growth
promotion. The plant growth promotion
potential of the bacterial strain dealt in this
study requires further evaluation and
va
lidation before its use as bio-
inoculants
in the
drought
agro ecosystems, where
alkali is a major determinant of plant and
microbial activity. The selection of native
functional plant growth promoting
microorganisms is a mandatory step for
reducing the use of energy intensive
chemical fertilisers. The strain reported in
this study seems to be an ideal candidate
for promotion as bio- inoculants, due to its
drought tolerance and multiple abilities of
plant growth promotion traits.
Acknowledgments
The authors are grateful to the Division of
Microbiology, Indian Agricultural
Research Institute (IARI), New Delhi and
Department of Biotechnology (DBT),
Ministry of Science and Technology for
providing the facilities and financial
support, to undertake the investi
gations.
References
Bakker, A. W. and Schippers B. 1987.
Microbial cyanide production in the
rhizosphere in relation to potato yield
reduction and Pseudomonas SPP-
mediated plant growth-
stimulation.
Soil Biol Biochem. 19 (4):451
-457.
Bric, J. M., Bostock R. M. and
Silverstone S. E. 1991. Rapid in situ
assay for indoleacetic acid production
by bacteria immobilized on a
nitrocellulose membrane. Appl
Environ Microbiol. 57 (2):535
-538.
Brown, M. E. and Burlingham S. K. 1968.
Production of plant growth subst
ances
by
Azotobacter chroococcum. J Gen
Microbiol. 53 (1):135
-
144.
Cappucino, J. C. and Sherman, N. 1992.
In: Microbiology: A Laboratory
Manual. 3rd ed. New York:
Benjamin/Cumming Pub. Co. (NH3
test).
Chaiharn, M. and Lumyong S. 2011.
Screening and Optimization of Indole-
3-Acetic Acid Production and
Phosphate Solubilization from
Rhizobacteria Aimed at Improving
Plant Growth. Curr Microbiol. 62
(1):173
-
181.
Conn, V. M. and Franco C. M. 2004.
Effect of microbial inoculants on the
indigenous actinobacterial endophyte
population in the roots of wheat as
determined by terminal restriction
fragment length polymorphism. Appl
Environ Microbiol. 70 (11):6407-
6413.
Coombs, J. T. and Franco C. M. 2003.
Isolation and identification of
actinobacteria from surface-
sterilized
wheat roots. Appl Environ Microbiol.
69 (9):5603
-5608.
Dobereiner, J., Urquiaga S. and Boddey
R. M. 1996. Alternatives for nitrogen
nutrition of crops in tropical
agriculture. In: Nitrogen Economy in
Tropical Soils. Springer, pp 338
-346.
Fels
enstein, J. 1981. Evolutionary trees
from DNA sequences: a maximum
likelihood approach. J Mol Evol. 17
(6):368
-376.
Glick, B., Patten C., Holguin G. and
Penrose D. 1999. Overview of plant
growth
-promoting bacteria.
Biochemical and Genetic Mechanisms
Int.J.Curr.Microbiol.App.Sci
(201
4)
3(5):
432
-
447
446
Used by Plant Growth Promoting
Bacteria".1
-13.
Goldstein, A. H. 1995. Recent progress in
understanding the molecular genetics
and biochemistry of calcium phosphate
solubilization by Gram negative
bacteria. Biol Agric Hortic
. 12 (2):185
-
193.
Holland, M. A. and Polacco J. C. 1994.
PPFMs and other covert contaminants:
is there more to plant physiology than
just plant? Annual Rev Plant Biol. 45
(1):197
-209.
Hu, X., Chen J. and Guo J. 2006. Two
Phosphate
- and Potassium-
solubilizing
Bacteria Isolated from Tianmu
Mou
ntain, Zhejiang, China. World J
Microbiol Biotechnol. 22 (9):983
-
990.
Ivanova, E., Doronina N. and Trotsenko
Y. A. 2001. Aerobic methylobacteria
are capable of synthesizing auxins.
Microbiology. 70 (4):392-397.
Jacobson, C. B., Pasternak J. and Glick B.
R. 1994. Partial purification and
characterization of 1-
aminocyclopropane
-1-carboxylate
deaminase from the plant growth
promoting rhizobacterium
Pseudomonas putida GR12-2. Can J
Microbiol. 40 (12):1019
-1025.
Jha, P. and Kumar A. 2009.
Characterization of novel plant growth
promoting endophytic bacterium
Achromobacter xylosoxidans from
wheat plant. Microb Ecol. 58 (1):179-
188.
Kumar, M., Yadav A. N., Tiwari R.,
Prasanna R. and Saxena A. K. 2013.
Deciphering the diversity of culturable
thermotolerant bacteria from
Manikaran hot springs. Ann
Microbiol.1
-11. doi:10.1007/s13213-
013-0709-7
Lavania, M., Chauhan P., Chauhan S. V.
S., Singh H. and Nautiyal C. 2006.
Induction of Plant Defense Enzymes
and Phenolics by Treatment With Plant
Growth Promoting Rhizobact
eria
Serratia marcescens NBRI1213. Curr
Microbiol. 52 (5):363
-
368.
Meena, K. K., Kumar M., Kalyuzhnaya M.
G., Yandigeri M. S., Singh D. P.,
Saxena A. K. and Arora D. K. 2012.
Epiphytic pink-
pigmented
methylotrophic bacteria enhance
germination and seedling growth of
wheat (Triticum aestivum) by
producing phytohormone. Antonie
Van Leeuwenhoek. 101 (4):777-786.
Nei, M. and Li W.
-
H. 1979. Mathematical
model for studying genetic variation in
terms of restriction endonucleases.
Proce Nat Acad Sci. 76 (10):5269-
5273.
Pandey, S., Singh S., Yadav A. N., Nain L.
and Saxena A. K. 2013. Phylogenetic
Diversity and Characterization of
Novel and Efficient Cellulase
Producing Bacterial Isolates from
Various Extreme Environments. Biosci
Biotechnol Biochem. 77 (7):1474-
1480.
Patten, C. L. and Glick B. R. 2002. Role
of
Pseudomonas putida indoleacetic
acid in development of the host plant
root system. Appl Environ Microbiol.
68 (8):3795
-3801.
Paul, D. and Nair S. 2008. Stress
adaptations in a plant growth
promoting rhizobacterium (PGPR)
with increasing salinity in the coastal
agricultural soils. J Basic Microbiol.
48 (5):378
-
384.
Pikovskaya, R. 1948. Mobilization of
phosphorus in soil in connection with
vital activity of some microbial
species. Mikrobiologiya. 17:362-370.
Rico, A., Ortiz Barredo A., Ritter E. and
Murillo J. 2004. Genetic
characterization of Erwinia amylovora
strains by amplified fragment length
Int.J.Curr.Microbiol.App.Sci
(201
4)
3(5):
432
-
447
447
polymorphism. J Appl Microbiol. 96
(2):302
-310.
Saitou, N. and Nei M. 1987. The
neighbor
-joining method: a new
method for reconstructing
phylogenetic trees. Mol Biol Evol. 4
(4):406
-425.
Sandhya, V., Ali S. Z., Venkateswarlu B.,
Reddy G. and Grover M. 2010. Effect
of osmotic stress on plant growth
promoting Pseudomonas
spp. Arch
Microbiol. 192 (10):867
-876.
Saravan
an, V. S., Subramoniam S. R. and
Raj S. A. 2004. Assessing in vitro
solubilization potential of different
zinc solubilizing bacterial (zsb)
isolates. Brazilian J Microbiol. 35 (1-
2):121
-125.
Schwyn, B. and Neilands J. 1987.
Universal chemical assay for t
he
detection and determination of
siderophores. Anal Biochem. 160
(1):47
-56.
Streptomyces, M. 2005. Isolation and
characterization of an endophytic
bacterium related to
Rhizobium
/Agrobacterium from wheat
(Triticum aestivum L.) roots. Curr Sci.
89 (4):608.
Tamura, K., Dudley J., Nei M. and Kumar
S. 2007. MEGA4: Molecular
Evolutionary Genetics Analysis
(MEGA) Software Version 4.0. Mol
Biol Evol. 24 (8):1596
-
1599.
Tilak, K., Ranganayaki N., Pal K., De R.,
Saxena A., Nautiyal C. S., Mittal S.,
Tripathi A. and Johri B. 2005.
Diversity of plant growth and soil
health supporting bacteria. Curr Sci. 89
(1):136
-150.
Verma, P., Yadav A. N., Kazy S. K.,
Saxena A. K. and Suman A. 2013.
Elucidating the diversity and plant
growth promoting attributes of wheat
(
Triticu
m aestivum) associated
acidotolerant bacteria from southern
hills zone of India. Natl J Life Sci. 10
(2):219
-226.
Wellner, S., Lodders N. and Kämpfer P.
2011. Diversity and biogeography of
selected phyllosphere bacteria with
special emphasis on
Methylobacterium
spp. Syst Appl
Microbiol. 34 (8):621
-
630.
Xie, H., Pasternak J. J. and Glick B. R.
1996. Isolation and Characterization of
Mutants of the Plant Growth-
Promoting Rhizobacterium
Pseudomonas putida GR12-2 That
Overproduce Indoleacetic Acid.
Curr
Mic
robiol. 32 (2):67
-71.
Yadav, A. N., Verma P., Kumar M., Pal K.
K., Dey R., Gupta A., Padaria J. C.,
Gujar G. T., Kumar S., Suman A.,
Prasanna R. and Saxena A. K. 2014.
Diversity and phylogenetic profiling of
niche specific Bacilli from extreme
environmen
ts of India. Ann
Microbiol.1
-20. Doi: 10.1007/s13213-
014-0897-9.
Yadav, S., Yadav S., Kaushik R., Saxena
A. K. and Arora D. K. 2013. Genetic
and functional diversity of fluorescent
Pseudomonas from rhizospheric soils
of wheat crop. J Basic Microbiol.1-13.
doi:
10.1002/jobm.201200384
Zinniel, D. K., Lambrecht P., Harris N. B.,
Feng Z., Kuczmarski D., Higley P.,
Ishimaru C. A., Arunakumari A.,
Barletta R. G. and Vidaver A. K.
2002. Isolation and characterization of
endophytic colonizing bacteria from
agrono
mic crops and prairie plants.
Appl Environ Microbiol. 68 (5):2198-
2208.
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