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ORIGINAL PAPER
Alleviation of drought stress effects in sunflower seedlings
by the exopolysaccharides producing Pseudomonas putida
strain GAP-P45
V. Sandhya &Ali SK. Z. &Minakshi Grover &
Gopal Reddy &B. Venkateswarlu
Received: 9 April 2009 / Revised: 13 August 2009 / Accepted: 17 August 2009 / Published online: 4 September 2009
#Springer-Verlag 2009
Abstract Production of exopolysaccharides (EPS) can be
used as a criteria for the isolation of stress tolerant
microorganisms. In the present study, EPS-producing
fluorescent pseudomonads were isolated from alfisols,
vertisols, inseptisols, oxisols, and aridisols of different
semiarid millet growing regions of India and were screened
in vitro for drought tolerance in trypticase soy broth
supplemented with different concentrations of polyethylene
glycol (PEG6000). Out of the total 81 isolates, 26 could
tolerate maximum level of stress (−0.73 MPa) and were
monitored for the amount of EPS produced under maxi-
mum level of water stress. The strain GAP-P45, isolated
from alfisol of sunflower rhizosphere, showed the highest
level of EPS production under water stress conditions, was
identified as Pseudomonas putida on the basis of 16S
rDNA sequence analysis, and was used as seed treatment to
study its effect in alleviating drought stress effects in
sunflower seedlings. Inoculation of Pseudomonas sp. strain
GAP-P45 increased the survival, plant biomass, and root
adhering soil/root tissue ratio of sunflower seedlings
subjected to drought stress. The inoculated bacteria could
efficiently colonize the root adhering soil and rhizoplane
and increase the percentage of stable soil aggregates.
Scanning electron microscope studies showed the formation
of biofilm of inoculated bacteria on the root surface and
this, along with a better soil structure, might have protected
the plants from the water stress.
Keywords Pseudomonas putida GAP-P45 .
Exopolysaccharide .Drought stress .Soil aggregate stabi lity .
Biofilm
Introduction
Drought stress is one of the major agricultural problems
limiting crop productivity in most of the arid and semiarid
regions of the world. The water potential of rhizosphere soil
is a key parameter that determines the availability of water,
oxygen, and nutrients to plants and microorganisms
(Postma et al. 1989; Blum and Johnson 1992; Van Gestel
et al. 1993). The complex and dynamic interactions among
microorganisms, roots, soil, and water in the rhizosphere
induce changes in physico-chemical and structural proper-
ties (Haynes and Swift 1990; Tisdall and Oades 1980)of
the soil. Microbial polysaccharides can bind soil particles to
form microaggregates (<250 μm diameter) and macro-
aggregates (>250 μm diameter; Oades 1993; Tisdall and
Oades 1982; Edwards and Bremmer 1967). Plant roots and
fungal hyphae fit in the pores between microaggregates
and thus stabilize macroaggregates (Oades and Waters
1991). Maintenance of soil structure is an important feature
of sustainable agriculture because it impacts a range of
processes influencing crop yield. Drought stress can
make physico-chemical and biological properties of soil
unsuitable for soil microbial activity and crop yield. Water
availability controls the production and consumption of
protein and polysaccharides by the bacteria (Roberson and
Firestone 1992) and thus indirectly influences soil structure.
V. Sandhya :A. SK. Z. :M. Grover (*):B. Venkateswarlu
Central Research Institute for Dry land Agriculture,
Santoshnagar,
Hyderabad 500059, India
e-mail: minigt3@yahoo.co.in
G. Reddy
Department of Microbiology, Osmania University,
Hyderabad 500007, India
Biol Fertil Soils (2009) 46:17–26
DOI 10.1007/s00374-009-0401-z
Bacteria like Pseudomonas can survive under stress con-
ditions due to the production of exopolysaccharide (EPS),
which protects microorganisms from water stress by
enhancing water retention and by regulating the diffusion
of organic carbon sources (Hepper 1975; Wilkinson 1958;
Roberson and Firestone 1992; Chenu 1993; Chenu and
Roberson 1996). EPS also help the microorganisms to
irreversibly attach and colonize the roots due to involvement
of a network of fibrillar material that permanently connects
the bacteria to the root surface (Bashan and Holguin 1997).
Bashan et al. (2004) reported the role of polysaccharides
producing Azospirillum in soil aggregation. Concentration
and composition of microbial EPS dramatically changed
under stress conditions. Capsular material of Azospirillum
brasilense Sp245 was found to contain high molecular
weight carbohydrate complexes (lipopolysaccharide–protein
(LP) complex and polysaccharide–lipid (PL) complex)
that could be responsible for protection under extreme
conditions, like desiccation. Addition of these complexes
to a suspension of decapsulated cells of A. brasilense
Sp245 significantly enhanced survival under drought
stress (Konnova et al. 2001). The EPS released into soil
as capsular and slime materials by soil microbes can be
adsorbed by clay surfaces due to cation bridges, hydrogen
bonding, Van der Waals forces, and anion adsorption
mechanisms, thus forming a protective capsule around soil
aggregates (Tisdall and Oades 1982). Plants treated with
EPS-producing bacteria display increased resistance to
water stress (Bensalim et al. 1998).Alamietal.(2000)
observed a significant increase in root adhering soil per
root tissue (RAS/RT) ratio in sunflower rhizosphere
inoculated with the EPS-producing rhizobial strain
YAS34 under drought conditions. Similar results were
obtained with wheat plantlets inoculated with Paenibacillus
polymyxa (Gouzou et al. 1993)andPantoea agglomerans
(Amellal et al. 1998) under salt stress. Hartel and Alexander
(1986) observed a significant correlation between the amount
of EPS produced by cowpea Bradyrhizobium strains and
their desiccation tolerance. Probably, it may possible to
alleviate drought stress in the plants by increasing the
population density of EPS-producing bacteria in the root
zone.
In the rain-fed agriculture systems, moisture stress in soil
is a major factor limiting crop production. Therefore, in the
present investigation, an attempt was made to isolate
drought tolerant Pseudomonas strains from cropped soils
of different arid and semiarid zones representing rain-fed
cropping system of India. An efficient EPS-producing,
drought tolerant Pseudomonas putida strain GAP-P45 was
characterized using biochemical and molecular approaches.
The effect of GAP-P45 inoculation on soil aggregation and
growth of sunflower seedlings under drought stress con-
ditions was studied.
Materials and methods
Isolation and screening
Pseudomonads were isolated from alfisols, vertisols, incep-
tisols, oxisols, and aridisols collected from rhizospheres of
millets and sunflower plants grown under 25 semiarid
locations across India. The plants were uprooted with
attached soil, brought to the lab under refrigerated
conditions, and immediately processed. Excessive soil from
the roots was removed by gentle shaking (Gouzou et al.
1993), and RAS was carefully collected and used for
isolation of fluorescent pseudomonads by serial dilution
method using King’s B (proteose peptone, 10 g; casein
enzyme hydrolysate, 10 g; K
2
HPO
4
, 1.5 g; MgSO
4
, 1.5 g;
and Agar, 15 g; per liter) as selective medium. The isolates
able to produce mucoid growth on King’s B medium after
incubation at 28±2°C for 48–72 h were stained with Indian
ink to check the presence of capsular material (Fett et al.
1989) and further screened for their ability to survive under
drought stress.
Bacterial growth under water stress
Trypticase soya broth (TSB) with different water potentials
(−0.05, −0.15, −0.30, −0.49, and −0.73 MPa) was prepared
by adding appropriate concentrations of polyethylene
glycol (PEG6000) (Michel and Kaufmann 1973) and was
inoculated with 1% of overnight raised cultures of the
bacterial isolates in TSB. Three replicates of each isolate
and each concentration were prepared. After incubation at
28°C under shaking conditions (120 rpm) for 24 h, growth
was estimated by measuring the optical density at 600 nm
using a spectrophotometer (Thermospectronic, 336002,
USA). The growth of the isolates at various stress levels
was recorded.
Extraction and purification of exopolysaccharides
The cultures able to grow at maximum stress level were
analyzed for their ability to produce EPS (Fett et al. 1986,
1989) under no stress and maximum stress level
(−0.73 MPa). Exopolysaccharide was extracted from
3-day-old cultures raised in TSB (25% PEG was added to
TSB for inducing stress). The culture was centrifuged at
20,000×g for 25 min and the supernatant was collected.
Highly viscous cultures were diluted with 0.85% KCL
before centrifugation. The pellet was washed twice with
0.85% KCl to completely extract EPS. The possible
extraction of intracellular polysaccharides was ruled out
by testing the presence of DNA in the supernatant by DPA
reagent (Burton 1956). Concentration of protein in the
supernatant was estimated by Folin’s reagent (Lowery et al.
18 Biol Fertil Soils (2009) 46:17–26
1951). Then, the supernatant was filtered through 0.45 μm
nitrocellulose membrane and dialysed extensively against
water at 4°C. The dialysate was centrifuged (20,000 × g) for
25 min to remove any insoluble material and mixed with
3 v of ice-cold absolute alcohol and kept overnight at 4°C.
The precipitated EPS obtained by centrifugation (10,000 × g
for 15 min) was suspended in water and further purified by
repeating the dialysis and precipitation steps. Total carbo-
hydrate content in the precipitated EPS was determined
according to Dubois et al. (1956). The isolate GAP-P45
showing maximum production of EPS under moisture
stress was selected for further studies
Acid hydrolysis of exopolysaccharides and thin-layer
chromatography
The precipitated EPS of isolate GAP-P45 was hydrolyzed
with 2 v of 2.5 M H
2
SO
4
at 100°C for 1 h, then the solution
was neutralized with 1 M sodium carbonate and applied to
the silica gel plates (Silica gel 60F 254; Merck) in a thin-
layer chromatography chamber using n-butanol: acetic acid:
water (4:1:5v/v) as the mobile phase at room temperature.
The plate was dried, sprayed with alkaline potassium
permanganate, and incubated at 100°C for 10 min. The Rf
values of colored spots were measured and compared with
those of standard carbohydrates (glucose, mannose, fructose,
mannitol, arabinose, xylose, rhamnose, raffinose, galactose,
and glucuronic acid; Horborne 1976).
Biochemical and molecular characterization of strain
GAP-P45
The GAP-P45 strain was characterized for Gram staining,
capsule formation, oxidase activity, catalase activity, urease
activity, gelatin hydrolysis, starch hydrolysis, citrate utili-
zation, production of H
2
S, production of indole, and acetyl
methyl carbinol and fermentation of glucose, sucrose,
trehalose, maltose, mannitol, xylose, fructose, galactose,
mannose, rhamnose, and sorbitol according to Holt et al.
(1994). Antibiotic resistance profile of the strain GAP-P45
was screened by testing resistance of the isolate to various
antibiotics such as ampicillin, amoxycillin, bacitracin,
cloxacillin, chloramphenicol, carbencillin, ciprofloxacin,
erythromycin, gentamycin, kanamycin, methicillin, nalidix-
icacid, polymyxin B, penicillin G, rifampicin, streptomycin,
tetracycline, trimethoprim, and vancomycin on solid medium
using antibiotic discs of different concentrations (Himedia,
India; Lalucat et al. 2006).
For molecular characterization, bacterial genomic DNA
was isolated (Chen and Kuo 1993)andsubjectedto
polymerase chain reaction (PCR) for amplification of 16S
rRNA gene using universal forward (5′-AGAGTTT
GATCCTGGCTCAG-3′) and reverse (5′-AAGGAGGT
GATCCAGCCGCA-3′) primers under standard conditions
(initial denaturation at 94°C for 5 min, 30 cycles of
denaturation at 94°C for 1 min, annealing at 50°C for
40 s, extension at 72°C for 90 s, and final extension at 72°C
for 7 min). The PCR (approximately 1.5 kb) product was
purified and sequenced (Oscimum Bio Solutions, India).
The partial 16S rDNA sequence was compared with the
sequences available in the GenBank, EMBL, and DJB
databases using the gapped BLASTN 2.2.21 program
through the National Center for Biotechnology Information
server and submitted to GenBank. Identification at the
species level was defined as a 16S rDNA sequence
similarity of ≥99% with that of the prototype strain
sequence in GenBank; identification at the genus level
was defined as a 16S rDNA sequence similarity of ≥97%
with that of the prototype strain sequence in GenBank. A
failure to identify was defined as a 16S rDNA sequence
similarity score of lower than 97% with those deposited in
GenBank at the time of analysis. The strain GAP-P45 was
also deposited at the National Bureau of Agriculturally
Important Microorganisms, Mau, Utter Pradesh, India.
Plant growth promoting properties of GAP-P45
The isolate GAP-P45 was tested in vitro for plant growth
promoting properties. For testing ammonia production,
culture was raised in 10 ml of peptone water at 28°C for
4 days, and 1 ml of Nesseler’sreagentwasadded.
Development of yellow to brown color indicated produc-
tion of ammonia (Dey et al. 2004). For siderophore
production, 1 µl of overnight raised culture in Luria broth
(LB) was spotted on Chrome Azurol S agar plates and
incubated at 28°C for 48–72 h. Plates were observed for the
appearance of orange halo around the bacterial colony
(Schwyn and Neilands 1987). For hydrogen cyanide (HCN)
production, the culture was streaked on King’s B medium
amended with 4.4 g l
−l
of glycine and Whatman number 1
filter paper disc soaked in 0.5% picric acid (in 2% sodium
carbonate) was placed in the lid of Petri plate. The plates
were sealed with parafilm and incubated at 28°C for 4 days
for the development of deep orange color (Bakker and
Schipper 1987). The method of Gordon and Weber (1951)
was followed for the estimation indole acetic acid (IAA).
One milliliter of the broth culture, raised in LB (amended
with 5 mM tryptophan), was centrifuged (10,000 g for
20 min), and supernatant was carefully decanted in a
separate test tube; 4 ml of Salkowsky reagent were added
to1 ml of supernatant and then the mixture was incubated
for 1 h at room temperature for the development of pink
color. After incubation, the absorbance was read at 530 nm.
Concentration of the proteins in the pellet was determined
(Lowery et al. 1951), and the amount of IAA produced was
expressed per milligram cell protein. For the estimation of
Biol Fertil Soils (2009) 46:17–26 19
gibberellic acid, 2 ml of zinc acetate were added followed
by 2 ml of potassium ferrocyanide to 15 ml of the culture
supernatant raised in minimal media. After centrifugation at
low speed for 15 min, 5 ml of the supernatant was taken in
a test tube, and 5 ml of 30% HCL were added followed by
incubation at 20°C for 75 min; then the absorbance was
read at 254 nm (Holbrook et al. 1961). For studying
phosphate solubilization, 5 µl of overnight raised culture
was spotted on Pikovskaya’s agar plates containing 2% tri-
calcium phosphate. The plates were incubated at 28°C for
24–48 h and observed for the appearance of the solubili-
zation zone around the bacterial colonies. For quantitative
analysis, 5 ml of NBRI-BBP medium (Mehta and Nautiyal
2001) in 30 ml test tubes were inoculated in triplicates with
50 µl of bacterial culture (2× 10
9
cfu/ml). Test tubes were
incubated for 7 days at 28°C on incubator shaker at
180 rpm. The cells were harvested by centrifugation at
2,655×g for 10 min, and the supernatant thus obtained was
analyzed for the concentration of unsolubilized phosphate
(Fiske and Subbarow 1925). Asymbiotic nitrogen fixation
was determined by growing the culture in nitrogen-free
malate medium according to Döbereiner and Day (1976).
Plant growth under drought stress
The Pseudomonas sp. strain GAP-P45, isolated from
sunflower rhizosphere grown in alfisol of Gunegal Re-
search Farm (GRF), Central Research Institute for Dryland
Agriculture (CRIDA), Hyderabad, India was an efficient
EPS producer under drought stress (−0.73 MPa) and
possessed plant growth properties (Table 1). Therefore, it
was selected for inoculation of sunflower plants under
drought stress conditions. Soil was collected from homo-
geneous horizon (0–20 cm) of GRF, CRIDA, a semiarid
region under rain-fed production system. The soil was air-
dried and sieved (<2 mm) before being analyzed for the
physico-chemical properties. The soil contained 71% sand,
3% silt, and 26% clay with 1.60 Mg m
−3
bulk density,
39.9% total porosity, and 37.9% water holding capacity; it
had pH 7.0 and electrical conductivity of 0.103 ms. Organic
C, total N and total P content of soil were, 0.62, 0.12, and
0.05 g/kg, respectively. Soil water content, determined by
drying the initially saturated soil at different matric
potentials by pressure plate apparatus (Santra Barbara,
CA, USA), was 16.5% (−0.3 MPa). Seeds of sunflower
(var. Sunbred) were surface sterilized with 0.1% HgCl
2
and
70% ethanol, washed with sterile distilled water, and coated
with talc based formulation (10
8
cells/g) of GAP-P45 using
1% carboxy methyl cellulose as adhesive. For the control
treatment, the seeds were treated with plain talc. The coated
seeds were shade dried and sown in plastic cups (surface
sterilized) filled with 950 g of sterile soil (sterilized for
three consecutive days). Both inoculated and uninoculated
treatments were replicated twenty times, and each treatment
had three plants per pot. The pots were incubated in a
controlled environmental chamber at 28/18°C day/night
temperature and a 16/8 h light/dark cycle (350 μmol m
−2
s
−1
light intensity). The soil moisture was adjusted at 75% of
water holding capacity (WHC). Soil moisture (12.5% of dry
weight of soil) was maintained constant during the
experiment by daily sprinkling with sterile distilled water.
After 11 days of germination, water stress was induced
in ten out of 20 replicates by discontinuing watering. After
15 days of germination (after 4 days of water stress), the
seedlings were harvested, and soil moisture in the pots was
measured using a HH
2
moisture meter (Theta probe type
ML2X Delta-T-device, England).
Harvesting, determination of RAS, RAS/RT ratio,
and aggregate water stability
Twelve plantlets per treatment were sampled. Roots with
adhering soil were carefully separated from bulk soil by
gentle shaking. RAS was removed by washing roots in
distilled water, and its EPS content was estimated (Šafařík
and Šantrůčková 1992). Shoot and root dry mass was
recorded after drying the samples at 105°C, and RAS/RT
ratio was calculated. Water stability of RAS was determined
by the wet sieving method. Root system with adhering soil
was passed through a set of sieves (2, 1, 0.5, and 0.25 mm)
and immersed in water and shaken. Amounts of water
stable aggregates (>0.25 mm) were calculated by substract-
ing coarse sand and root fragments remaining on the sieve.
Oven-dried soil aggregates were transferred into dispersion
cups and stirred for 10 min with 10% sodium hexameta
phosphate to remove clay particles from microaggregates,
Plant growth promoting properties Non-stressed conditions Drought-stressed conditions
Ammonia production +++ ++
Hydrogen cyanide ++ +
Indole acetic acid 329.33 (±10.12) μgmg
−1
protein 171 (±2.60) μgmg
−1
protein
Gibberellins 382.7 (±2.1) μgmg
−1
protein 105.0 (±13.5) μgmg
−1
protein
P-solubilization 69.3 (±6.7) μgml
-−1
41.7 (±4.0) μgml
−1
Siderophore + +
Table 1 Plant growth promoting
properties of P. putida strain
GAP-P45 under non-stressed
and drought-stressed conditions
+, presence; +, fair; ++, good;
+++, excellent
20 Biol Fertil Soils (2009) 46:17–26
and aggregate stability and mean weight diameter were
recorded (Chaudhary and Kar 1972; Bartoli et al. 1991).
Relative water content (RWC) of leaves was determined by
recording fresh weight, saturated weight, and dry weight of
leaves (Teulat et al. 2003).
Enumeration of rhizobacteria
Total counts of inoculated strain GAP-P45 in bulk soil,
RAS, and rhizoplane were determined on the 15th day of
sowing using King’s B medium containing antibiotic
(trimethoprim and vancomycin, 30 μg each). The entire
root system with adhered soil was removed from the pot
and agitated gently, to get the bulk soil fraction. The RAS
samples were obtained as mentioned earlier. Serial dilutions
of roots and soil suspensions were prepared, and appropriate
dilutions were plated on King’s B medium supplemented
with trimethoprim (30 μg/ml) and vancomycin (30 μg/ml).
After incubation at 28°C for 24–48 h, fluorescent colonies
were counted.
Colonization and biofilm formation by GAP-P45
After harvesting, the root samples were fixed in 2.5%
Gluteraldehyde in 0.05 M phosphate buffer (pH 7.2) for
24 h at 4°C and post fixed in 2% aqueous osmium
tetraoxide in the same buffer for 2 h. After the post
fixation, samples were dehydrated with alcohol and
mounted over the stubs with double-sided conductivity
tape. A thin layer of gold metal was applied over the
samples using an automated sputter coater (JEOL JFC-1600).
The samples were scanned under scanning electron micro-
scope (Model: JOEL-JSM 5600, JAPAN) at various magni-
fications (RUSKA Lab, Hyderabad, India; Bozzola and
Russell 1999). The root samples were also stained with
acridine orange (0.5% w/vin PBS) for 5 min at room
temperature and immediately observed under fluorescent
microscope using green filter (800 nm).
Statistical analysis
Comparisons between treatments were carried out by one-
way analysis of variance (ANOVA). Tukeys test was
applied after ANOVA for heterogeneity of variance.
Results
Isolation and screening
A total of 212 fluorescent pseudomonads were isolated
on King’s B media, of which, 81 showed mucoid growth
on King’sBmediaandthepresenceofcapsularmaterial
under microscope. These EPS-producing isolates were
screened for drought tolerance on solid media at varying
water potential. Isolates, which could tolerate higher
levels of drought stress, were used for EPS production
under both no stressed conditions as well as under
minimum water potential (−0.73 MPa). The strain GAP-
P45 produced a significant amount of EPS (4.06 mg mg
−1
protein) under minimum water potential (−0.73 Mpa) when
compared to other strains and also showed higher cell
viability at −0.73 Mpa.
Exopolysaccharide produced by GAP-P45 was acid
hydrolyzed, and component sugars were qualitatively
identified by thin-layer chromatography. The Rf values of
the yellow colored spots developed were recorded, com-
pared with those of standard sugars. The hydrolysate of
EPS of GAP-P45 strain produced spots on silica gel plates,
with Rf values of 0.18, 0.28, corresponding to glucose and
mannose, respectively, under no stress and spots with Rf
values of 0.172, 0.26, and 0.39, corresponding to
glucose, mannose, and rhamnose, respectively, under
stress conditions.
Biochemical and molecular characterization of the isolate
GAP-P45
Microscopic studies revealed strain GAP-P45 as Gram-
negative, motile, capsulated rods having pale yellow, entire,
convex, opaque, and mucoid colony morphology. The
isolate showed presence of oxidase activity, catalase
activity, urease activity, and could utilize citrate as carbon
source; it produced H
2
S and acid from glucose and
fructose. Production of acetyl methyl carbinol was not
observed. Based on biochemical characterization, the strain
GAP-P45 was identified as Pseudomonas sp. Molecular
characterization of the strain was done on the basis of 16S
rDNA gene sequence that showed 99% homology with that
of Pseudomonas putida in the existing database of National
Center of Bioinformatics. The sequence was submitted to
GenBank under the accession no GQ221267. The strain
GAP-P45 was also deposited at the National Bureau of
Agriculturally Important Microorganisms, Mau, Utter
Pradesh, India.
P. putida GAP-P45 strain showed resistance to amoxy-
cillin (25 µg/disc), cloxacillin (10 µg/disc), kanamycin
(30 µg/disc), methicillin (30 µg/disc), vancomycin (30 µg/
disc), penicillin (6 µg/disc), and trimethoprim (30 µg/disc).
The isolate GAP-P45 also exhibited plant growth promot-
ing properties (Table 1) like production of ammonia,
siderophore, HCN, IAA, gibberellic acid, and P-
solubilization both under no stress and drought stress
conditions; significant reduction in plant growth promoting
traits was observed under drought stress. The production of
IAA and gibberellic acid under no stress conditions was
Biol Fertil Soils (2009) 46:17–26 21
329.33 μgmg
−1
protein and 382.7 μgmg
−1
protein,
respectively, whereas under drought stress conditions, the
production was 171.0 μgmg
−1
protein and 105.0 μgmg
-1
protein, respectively. Phosphate solubilization was 69.3 and
41.7 ppm ml
−1
under no stress and stress conditions,
respectively.
Growth studies
Drought stress drastically affected the growth of sunflower
seedlings as reflected by stunted growth, less vigor, and
drying of leaves. After 15 days of germination, soil
moisture in pots subjected to drought stress was 8.2%
(49.6% of WHC) as compared to 12.37% (75% of WHC) in
pots without stress. Inoculation of EPS-producing P. putida
strain GAP-P45 significantly increased total root, shoot
length (Fig. 1a), and dry biomass in sunflower seedlings,
both under no stress and drought stress conditions, and the
effects of inoculation on root dry biomass were higher
(53.92% and 45.1% under no stress and drought stress
conditions, respectively) than on shoots (Fig. 1b). An
increase in total dry biomass by 64.6% and 23% due to
strain GAP-P45 inoculation was observed under drought
stress and no stress conditions, respectively. Leaves of
inoculated seedlings under drought stress had RWC
compared to that of uninoculated seedlings maintained
under no stress conditions.
There was a positive effect of inoculation on RAS/RT
ratio, which increased by 12% and 49.8% under no stress
and drought stress conditions, respectively, and the effect
was positively correlated with the content of water
insoluble saccharides in RAS (Table 2).The effect of P.
putida GAP-P45 on aggregation of rhizosphere soil was
assessed by determining the percentage of water stable
aggregates in RAS. Inoculation significantly enhanced
percentage of water stable aggregates (diameter >0.25 mm)
from 30± 2 to 51 ± 4% and from 28±1% to 67 ± 1% under no
stress and drought stress, respectively. Mean weight diameter
of soil aggregates was also significantly increased due to
GAP-P45 inoculation under both no stress and drought stress
conditions.
After 15 days of germination, the population of strain
GAP-P45 in bulk soil, RAS, and rhizosplane was 6 × 10
3
(±5.279), 6×10
6
(±8.085), 2×10
7
(±12.29) CFU g
−1
soil/
root under no stress and 4× 10
2
(±5.203), 5×10
5
(±5.657),
3×10
6
(±9.94) CFU g
−1
soil/root, respectively, under
drought stress conditions, respectively. The population of
uninoculated bulk soil, RAS, and rhizoplane was 4 × 10
2
(±6.129), 3×10
1
(±13.21), 3×10
1
(±16.01) CFU g
−1
soil/
root under no stress and 2× 10
1
(±3.110), 2× 10
1
(±6.112),
2×10
1
(±11.25) CFU g
-1
soil/root, respectively, under
drought stress conditions, respectively.
Scanning electron microscope studies also showed
bacterial colonization on the root surface both under normal
and drought stress conditions (Fig. 2) and revealed that the
strain GAP-P45 produced biofilm like communities, where
bacteria were connected together by an extracellular
polymeric matrix with micro colony formation under water
stress conditions. Staining of the roots with acridine orange
(DNA staining dye) further confirmed root surface coloni-
zation by the bacteria.
Discussion
The management of drought-affected soils is essential to
meet the ever increasing food demands. Introduction of
EPS-producing microorganisms in the drought-stressed
soils can alleviate stress in the crop plants. In the present
investigation of the 212 fluorescent pseudomonads
isolated from soils of different semiarid regions of the
country, only 81 isolates (38.2%) produced mucoid
0
5
10
15
20
25
30
NSUI NSI DSUI DSI
Root and shoot length (cm)
ROOT SHOOT
0
100
200
300
400
500
600
700
800
900
NSUI NSI DSUI DSI
Root and shoot dry biomass (mg)
ROOT SHOOT
a b
Fig. 1 Growth promotion of sunflower seedlings inoculated with P.
putida strain GAP-P45 ((a) root and shoot length; (b) root and shoot
dry biomass). NSUI non-stressed uninoculated, NSI non-stressed
inoculated, DSUI drought-stressed uninoculated, DSI drought-
stressed inoculated. Values with different letters are significantly
different at P<0.05 in all the treatments
22 Biol Fertil Soils (2009) 46:17–26
growth. Of these 81 isolates, 26 could tolerate maximum
level of drought stress (−0.73 Mpa). The EPS production
of these selected isolates was higher under stressed than
under no stress conditions, and it increased by increasing
stress level, indicating that EPS production in bacteria
occurs as a response to the stress (Roberson and
Firestone 1992).
Role of capsular material has been suggested in the
protection of A. brasilense Sp245 cells against desiccation
(Konnova et al. 2001). Probably EPS can provide a
Table 2 Effect of Pseudomonas sp. strain GAP-P45 inoculation on soil structure and physiology of sunflower seedlings
Treatments Root adhering soil dry weight per
root tissue ratio (mg/mg)
Exopolysaccharide
(mg/plant)
Aggregate
stability (%)
Mean weight
diameter (mm)
Relative water
content (%)
Soil moisture
(%)
Non-stressed
uninoculated
18.23a (±0.49) 14.37a (±0.51) 30.00a (±2.00) 0.170a (±0.02) 53.70a (±1.60) 12.3a (±0.04)
Non-stressed
inoculated
20.60b (±0.52) 54.00b (±2.26) 51.33b (±4.50) 0.356b (±0.05) 60.20b (±1.06) 12.3ba (±0.04)
Drought-stressed
uninoculated
24.80c (±0.32) 15.67ca (±0.30) 28.40ca (±0.69) 0.140ca (±0.02) 40.60c (±0.85) 8.16c (±0.65)
Drought-stressed
inoculated
10.36d (±0.76) 63.30db (±9.95) 70.80d (±0.80) 0.389db (±0.09) 51.30da (±1.17) 8.20dc (±0.25)
Mean values with different letters are significantly different at 5% probability level (mean±standard deviation)
Fig. 2 Scanning electron microscopic micrographs of sunflower roots colonized by P. putida strain GAP-P45. (a) inoculated, non-stressed (b)
uninoculated, non-stressed (c) inoculated, drought-stressed (d) uninoculated, drought-stressed
Biol Fertil Soils (2009) 46:17–26 23
microenvironment that holds water and dries more slowly
than the surrounding microenvironment, thus protecting
bacteria from drying and fluctuations in water potential
(Hepper 1975; Wilkinson 1958). Isolate GAP-P45 pro-
duced significant amount of EPS under minimum water
potential, exhibited plant growth promoting properties like
P-solubilization, produced of ammonia, siderophore, HCN,
IAA, and gibberellic acid both under no stressed and
drought-stressed conditions (Table 1) and was identified as
P. putida on the basis of 16S rRNA gene sequence analysis.
Seed inoculation of sunflower with the P. putida GAP-P45
could help the plants in tolerating drought stress as
indicated by significantly high root and shoot dry biomass
(Fig 1b), the RAS/RT ratio, the aggregate stability, and
mean weight diameter of the inoculated plants under
stressed conditions (Table 2). Plants with higher root
biomass had higher microbial biomass and percent aggre-
gation in RAS (Haynes and Francis 1993). Production of
EPS by bacteria improved RAS permeability by increasing
soil aggregation and maintaining higher water potential
around the roots; in this way, there was an increase in the
uptake of nutrients by plant, with an increase in plant
growth; in addition, the bacteria protected the seedlings
from drought stress (Alami et al. 2000; Miller and Wood
1996). A high insoluble saccharide content of RAS of the
inoculated seedlings indicated an enhanced EPS synthesis
in the root zone. There was also an increased mass of soil
aggregated around roots of the inoculated seedlings with a
highly significant positive correlation (P<0.01) between
water insoluble saccharides and RAS/RT ratio; these data
indicated the role of bacterial EPS in aggregating the soil
around roots (Watt et al. 1993; Alami et al. 2000; Bezzate et
al. 2000). Higher EPS content and better aggregation of
RAS could help the plants to take up a higher volume of
water and nutrients from rhizosphere soil (Miller and Wood
1996), resulting in a better growth of plants, and also, this
was useful to counteract the negative effects of drought
stress (Munns 2002). Probably other factors like mechanical
impedance (the axial root pressure exerted per unit area) and
gaseous and moisture contents of the RAS-root association
could also have influenced the plant growth and crop yield of
the inoculated seedlings. Under dry conditions, the increased
root biomass and the rhizobacterial population probably
increased root and soil microbial respiration with influences
on the composition of soil atmosphere. All these RAS factors
can control and regulate growth and functioning of roots
(Mohr and Schopfer 1996;Clarkeetal.2003; Kuzyakov and
Larionova 2005; Wittenmayer and Merbach 2005). In
uninoculated seedlings, due to the absence of EPS-
producing bacterial populations, most of roots was
devoid of RAS and thus was more susceptible to stress
effect. Moreover, a higher population of EPS-producing
bacteria on roots of inoculated plants may have stimu-
lated root exudation (Tisdall 1994; Miller and Wood
1996; Wittenmayer and Merbach 2005), with stimulation
of growth of inoculated bacteria with higher EPS
production in the rhizosphere (Fischer et al. 2003).
Scanning electron microscope confirmed the colonization
of bacteria and biofilm formation on the surface of the
roots (Fig. 2). Most of the nutrients and water taken up by
the plants passes through the interfacial region, that is the
soil adhering strictly to plant roots (McCully and Canny
1988;Wattetal.1994).
Conclusion
The EPS-producing Pseudomonas strain GAP-P45 act as a
plant growth promoting rhizobacteria and can alleviate the
effect of drought stress in sunflower plants possibly through
improved soil structure and plant growth promoting
substances. The moisture sorption and colloidal stabiliza-
tion properties of EPS are important and should be
considered in combination with other factors like spread
of bacteria along the root system and physical properties of
RAS. Good soil structure in the rhizosphere could improve
growth of the seedlings, as mediated by efficient uptake of
nutrients and water.
Acknowledgements The authors are grateful to Indian Council of
Agricultural Research (ICAR), New Delhi, for providing the financial
assistance in the form of network project on “Application of
Microorganisms in Agriculture and Allied Sectors”(AMAAS).
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