Applied Soil Ecology 61 (2012) 264–272

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Applied Soil Ecology

journal homepage: www.elsevier.com/locate/apsoil

Effect of inoculation with plant growth-promoting bacteria (PGPB) on

amelioration of saline stress in maize (Zea mays)
Daniel Rojas-Tapias, Andrés Moreno-Galván, Sergio Pardo-Díaz, Melissa Obando, Diego Rivera,
Ruth Bonilla ∗
Laboratorio de Microbiología de Suelos, Centro de Biotecnología y Bioindustria (CBB), Corporación Colombiana de Investigación Agropecuaria, CORPOICA, Km. 14 vía Bogotá, Mosquera,
Colombia

a r t i c l e i n f o a b s t r a c t

Article history: Our objective was to evaluate the role of Azotobacter strains to protect maize plants against salt damage.
Received 23 June 2011 Four candidate Azotobacter strains were evaluated, and the two most tolerant to salinity (C5 and C9) were
Received in revised form selected for further studies. They were phylogenetically related to Azotobacter chroococcum based on their
30 September 2011
16S rDNA sequences. Strains were inoculated on maize roots growing in sterilized soil under different
Accepted 14 January 2012
salinity conditions (0, 2.93 and 5.85 g NaCl/kg soil). After 4 weeks plant biomass (length and weight),
ion uptake (Na+ , K+ , Ca2+ , Mg2+ ), chlorophyll content, and accumulation of proline and polyphenols were
Keywords:
evaluated. Strains C5 and C9 fixed nitrogen and solubilized phosphate regardless of NaCl concentration
Plant growth-promoting bacteria (PGPB)
Zea mays
in most cases, while auxins were synthesized by C5 only under conditions of salinity. In pot experiments,
Saline stress plant growth was promoted by bacterial inoculation only at 2.93 and 5.85 g NaCl/kg soil (P < 0.05). Bacte-
Azotobacter chroococcum ria improved Na+ exclusion and K+ uptake in maize, thereby increasing their K+ /Na+ ratio. Content of
Proline polyphenol and chlorophyll was enhanced by inoculation with strains C5 and C9. The concentration of
Polyphenols proline in leaves was increased by salinity, but was decreased when C5 and C9 were used as inoculants.
The present observations showed that strains C5 and C9 partially alleviated the saline stress in maize,
likely through the integration of several mechanisms that improve the plant response. Hence, the use of
nitrogen-fixing plant growth-promoting bacteria may represent an important biotechnological approach
to decrease the impact of salinity in crops.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction Currently, more than 800 million hectares of land throughout

the world are affected by levels of salt that could substantially
Salinity is one major limiting factor to plant growth and crop reduce crop productivity (Munns and Tester, 2008). Suboptimal
productivity (Allakhverdiev et al., 2000). In most saline soils, irrigation can result in further damage, caused by salinity in irriga-
sodium chloride is the predominant salt species, and its effect can tion waters, on several important agricultural crops. For instance,
be observed by decreased productivity or plant death (Munns and maize is considered to be a moderately salt-sensitive plant (Zörb
Tester, 2008). Soil salinity causes plant stress in two ways: (1) et al., 2004), and under irrigation, it can be subjected to salt tox-
making water uptake by the roots more difficult, and (2) caus- icity (Fu et al., 2010). Several strategies have been developed in
ing plant toxicity via accumulation of high salt concentrations in order to decrease the toxic effects caused by high salinity on plant
the plant (Munns and Tester, 2008). Several biochemical processes growth, including plant genetic engineering (Wang et al., 2003),
can be affected by salinity, including protein synthesis, photo- and recently the use of plant growth-promoting bacteria (PGPB)
synthesis, and lipid metabolism (Parida and Das, 2005). However, (Dimkpa et al., 2009).
most plants possess several mechanisms to decrease the negative PGPB are usually defined as microorganisms that can grow in,
effects of salinity including regulation and compartmentalization on, or around plant tissues, stimulating plant growth by a variety
of ions, synthesis of compatible solutes, induction of antioxidative of mechanisms (Vessey, 2003). These mechanisms and their effects
enzymes, induction of plant hormones, and changes in photosyn- can be classified as direct or indirect. The direct mechanisms are
thetic pathways (Cheeseman, 1988; Parida and Das, 2005). associated with an increase in availability of nutrients and include
biological nitrogen fixation (BNF) (Graham and Vance, 2000), phos-
phate solubilization and mineralization (Rodríguez et al., 2007),
∗ Corresponding author. Tel.: +57 1 4227300x1400. siderophore production (Neilands, 1993), and synthesis of plant
E-mail addresses: [email protected], hormones such as indole, cytokinins, or gibberellins (Costacurta
[email protected], [email protected] (R. Bonilla). and Vanderleyden, 1995). Utilization of PGPB has become a

0929-1393/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.apsoil.2012.01.006
 D. Rojas-Tapias et al. / Applied Soil Ecology 61 (2012) 264–272 265

promising alternative to alleviate plant stress caused by salinity (Fu a 25 ␮L PCR mixture contained 1× Taq DNA polymerase buffer
et al., 2010; Mayak et al., 2004; Shilev et al., 2010; Yao et al., 2010). (Invitrogen, USA), 2.5 mM MgCl2 , 0.2 mM of each deoxynucleoside
Pseudomonas fluorescens biotype F and P. fluorescens CECT 378T triphosphate, 25 pmol of each forward and reverse primers, 1 U of
increased fresh weight of sunflowers by more than 10% under saline DNA polymerase (Invitrogen, USA), and 50 ng of genomic DNA as
conditions (100 mM NaCl), and similarly improved the K+ /Na+ ratio template. Nearly complete 16S rDNA genes were amplified with the
(Shilev et al., 2010). Studies showed that inoculation with Azospiril- forward primer 27F (5′ -AGA GTT TGA TCC TGG CTC AG-3′ ) and the
lum spp. increased plant growth and the K+ /Na+ ratio of two maize reverse primer 1492R (5′ -GGT TAC CTT GTT ACG ACT T-3′ ) (Gauri
cultivars cv. 323 and cv. 324 (Hamdia et al., 2004). Moreover, Yao et al., 2009). The DNA was amplified with an iCycler thermocycler
et al. (2010) reported that inoculation with Pseudomonas putida Rs- (BioRad, USA) with the following program: 2 min of pre-heating
198 promoted cotton growth and germination under conditions of at 95 ◦ C, 35 cycles of 30 s of denaturation at 95 ◦ C, 30 s of primer
salt stress. annealing at 57 ◦ C, 2 min of elongation at 72 ◦ C, and 10 min of exten-
Hence, the main purposes of this research were: (1) to focus sion step at 72 ◦ C. Amplicon size was confirmed to be as expected by
on the identification of the Azotobacter sp. strains C5 and C9 with using agarose gel electrophoresis (1.5% w/v) in TAE buffer (0.04 M
known plant protection against salt stress, (2) to study the effect Tris acetate, 0.001 M EDTA) containing 1 ␮g mL−1 ethidium bro-
of PGPB on plant growth in the presence and absence of salt stress, mide. The amplified DNA was purified using the PureLinkTM Quick
and (3) to evaluate the influence of bacteria on uptake of ions (Na+ , Gel Extraction Kit (Invitrogen, USA) according to the manufacturer’s
K+ , Ca2+ , Mg2+ ) and accumulation of proline, total polyphenols, and instructions. Automated sequencing of the purified PCR products
chlorophyll in maize. was performed using the BigDye terminator cycle sequencing kit
and the ABI 310 DNA Sequencer (Applied Biosystems, Foster City,
2. Materials and methods CA) according to the manufacturer’s instructions. Partial sequences
obtained were matched against nucleotide sequences present in
2.1. Strains and culture conditions GenBank using the BLASTn program and deposited in the EMBL-
EBI/GenBank database.
In this study, four presumptive strains of Azotobacter sp.: C5,
C7, C8, and C9, were studied. These were previously selected by
their potential as biofertilizers (data not shown), and were provided 2.4. Plant growth-promoting (PGP) features
by Laboratorio de Microbiología de Suelos of Corpoica, Colombia.
Strains were isolated in Provincia de Ricaurte, Boyacá, Colombia Measurement of plant growth-promoting features was carried
(5◦ 38′ 02.69′′ N 73◦ 31′ 24.02′′ O, elevation 2142 m). The standard cul- out at 0, 2.93, and 5.85 g NaCl/L. Each experiment was performed
ture conditions for incubation were 28 ± 2 ◦ C and 150 rpm. Bacterial in triplicate at two different times.
maintenance utilized Ashby medium (in g/L: mannitol 10, K2 HPO4
0.2, MgSO4 ·7H2 O 0.2, NaCl 0.2, CaSO4 0.1, CaCO3 10.0, agar 15.0,
pH 7.5). Morphological characteristics, such as gram reaction, cell 2.4.1. Biological nitrogen fixation
shape, and cyst formation, were examined after incubation in Flasks (250 mL) containing 50 mL free-nitrogen Ashby medium
nitrogen-free Ashby culture medium for 3 days using an optic were inoculated with 25 ␮L of bacterial suspension adjusted to
microscope (Olympus, Japan). Pigmentation was also observed OD600 = 0.500 and incubated for 24 h at 30 ◦ C. Biological nitro-
after 3 days of incubation in the same culture media. For catalase gen fixation was measured by using a gas chromatograph (Perkin
assay, one bacterial colony and one drop of 3% hydrogen peroxide Elmer, USA) with flame ionization detector and a Poropak col-
(Merck, USA) were used, the appearance of bubbles was consid- umn N 200/300 Mesh of 6.0 ft and 3.0 mm diameter, according to
ered as positive. The API20NE kit (bioMérieux, France) was used Eckert et al. (2001). A calibration curve was determined by using
for the evaluation of hydrolysis of urea, esculine, and gelatine ethylene (chromatographic grade) as standard. Confirmation of
hydrolysis; utilization of glucose, arabinose, mannose, mannitol, N- nitrogen fixation capability was evaluated by amplifying the nifH
acetyl-glucosamine, maltose, caprate, malate, citrate, and phenyl gene according to the method described by Widmer et al. (1999).
acetate; and for nitrate reduction to nitrite. All tests were per-
formed in duplicate.
2.4.2. Bacterial phosphate solubilization
2.2. Effect of NaCl on bacterial growth For the evaluation of phosphate solubilization, the quantitative
phosphomolybdate method was employed. Each strain was grown
Tolerance of strains to NaCl was evaluated on Ashby modified on Pikovskaya broth (in g/L: glucose 10; (NH4 )2 SO4 0.5; MgSO4 0.1,
broth (in g/L: mannitol 10, K2 HPO4 0.2, MgSO4 ·7H2 O 0.2, NaCl KCl 0.2, yeast extract 0.05, Ca3 (PO4 )2 5.0); and incubated for 120 h
0.2, CaSO4 0.1), supplemented with increasing NaCl concentrations at 150 rpm (Pikovskaya, 1948). Five-hundred microliters of super-
ranging between 0 and 58.5 g/L. Flasks (125 mL) were incubated natant from each culture, including controls, were used for analysis
for 72 h at standard conditions. In addition, we studied the effect (Fiske and Subbarow, 1925). A calibration curve was determined by
of NaCl (0, 2.93, and 5.85 g/L) on growth kinetics of the C5 and C9 using K2 HPO4 (Merck, USA).
strains employing culture flask (250 mL) containing 50 mL of Ashby
modified broth. Flasks were inoculated with 500 ␮L of an overnight
culture adjusted to OD600 = 0.500, and incubated for 72 h at stan- 2.4.3. Indolic compound synthesis
dard conditions. The bacterial growth was monitored by measuring Indolic compounds were estimated using the colorimetric assay
optical density at 600 nm. based on the Salkowsky reagent using the PC reagent (12 g/L
FeCl3 in 7.9 M H2 SO4 ) (Glickmann and Dessaux, 1995). The cul-
2.3. Genetic characterization of strains ture medium used was K-lactate (Carreno-Lopez et al., 2000), and
the incubation was carried out for 72 h at 150 rpm in the dark. The
Bacterial DNA was extracted by using DNeasy Blood & Tissue reaction between the PC reagent and culture supernatant was per-
Kit (Qiagen, Germany) according to the manufacturer’s instruc- formed in a 1:1 ratio for 30 min in the dark. Indolic compounds
tions. Genomic DNA extracted was diluted in sterile Milli-Q water were spectrophotometrically determined at 540 nm. A calibration
before conducting PCR analysis under the following conditions: curve was determined using indol-3-acetic acid pure (Merck, USA).
266 D. Rojas-Tapias et al. / Applied Soil Ecology 61 (2012) 264–272

Table 1
Characteristics of soil used for pot experiments.

Parameter Value Parameter Value

pH 5.95 ± 0.07 Sodium (cmol/kg) 0.39 ± 0.15

Organic matter (%) 15.15 ± 0.21 Effective cationic interchange capacity 8.26 ± 1.81
Phosphorus (ppm) 13.6 ± 0.70 Electric conductivity (dS/m) 0.69 ± 0.2
Sulphur (ppm) 12.6 ± 0.70 Minor elements
Interchangeable cations Iron (ppm) 134.5 ± 16.2
Calcium (cmol/kg) 4.72 ± 0.69 Copper (ppm) 1.85 ± 0.07
Magnesium (cmol/kg) 1.11 ± 0.35 Manganese (ppm) 17.30 ± 0.01
Potassium (cmol/kg) 1.95 ± 0.75 Zinc (ppm) 3.45 ± 0.9

± shows standard deviation.

2.5. Effects of NaCl and bacterial inoculation on maize growth 2.8. Estimation of proline in plant

Pot experiments were conducted in order to evaluate the effect Proline content was estimated according to Bates et al. (1973).
of NaCl and bacterial inoculation on growth of Zea mays. The exper- Briefly, 0.5 g fresh leaves were frozen in liquid nitrogen, homog-
imental design was a full factorial design with six replicates per enized with 3% sulfosalicylic acid (Fisher Scientific, USA), and
treatment. Pots containing 400 g of dry-sterilized soil were supple- immediately centrifuged at 10,000 × g for 5 min. One milliliter of
mented to reach 0, 2.93 and 5.85 g NaCl/kg soil, which was prepared supernatant was taken for analysis. Absorbance was measured at
by adding 0, 1.078 and 2.248 g NaCl dissolved in 100 mL water. The 520 nm and the calibration curve was determined using pure l-
treatment without exogenous addition of NaCl was considered as proline (Sigma–Aldrich, USA) as standard.
0 g NaCl/kg soil concentration. Characteristics of the soil without
added salt are listed in Table 1. For measuring electrical conduc- 2.9. Estimation of total polyphenol content
tivity, 30 g dry soil was mixed with 20 mL deionized water and
shaken for 1 h. Then, the soil extract was filtered, and conductiv- Total polyphenols were analyzed as described Parida et al.
ity was measured using a conductivimeter (Thermo Corporation, (2002). For this, 0.5 g fresh leaves were frozen in liquid nitrogen and
USA). Maize seeds Var. ICA-508 were disinfected by soaking in homogenized in 5.0 mL of 80% ethanol by using a chilled pestle and
30% hydrogen peroxide and 70% ethanol (1:1) for 10 min, and fol- mortar, with subsequent centrifugation at 10,000 × g for 10 min.
lowed by rinsing several times in sterilized distilled water. The The supernatant was conserved and the residue re-extracted with
seeds were then pre-germinated in sterilized peat at room condi- 2.5 mL of 80% ethanol and centrifuged again. The supernatant was
tions for five days. For inoculum preparation, bacteria were growth pooled and evaporated to dryness. The residue was dissolved in
in nutrient broth (Merck, USA) for 24 h at 28 ◦ C, rinsed twice, and 5.0 mL of distilled and deionized water and analyzed. Supernatants
finally resuspended to the same initial volume using 0.03 M MgSO4 . were taken for analysis. Absorbance was measured at 650 nm. A
Roots of seedlings, with the same size, were submerged three times standard curve was determined using pure gallic acid (Merck, USA)
in bacterial suspension adjusted to OD600 = 1.000 and planted in as standard.
each pot supplemented or not with NaCl. Seedlings submerged
in sterilized water were used as a control. Plants were grown in 2.10. Statistical analysis
a growth chamber at a day/night temperature of 22/18 ◦ C with
120 ␮mol m−2 s−1 of light supplied for 16 h during daytime for 4- Statistical analyses were carried out by using the software
weeks. Finally, vegetative tissue was employed for the respective package SPSS version 17.0. Data were analyzed using Analysis of
analyses described below. To determine the dry weight, shoots Variance (ANOVA) and the HSD Tukey pairwise comparisons. Asso-
and roots were oven-dried separately at 60 ◦ C for 48 h (at con- ciations among characters were examined by simple correlation
stant weight). In addition, lengths of both shoots and roots were analysis. All tests were subjected to a 95% confidence limit.
recorded.
3. Results
2.6. Determination of chlorophyll
3.1. Effect of NaCl on bacterial growth
Photosynthetic pigment content of Zea mays leaves was esti-
mated by the method of Hiscox and Israelstam (1979), employing High levels of NaCl repressed bacterial growth, where strains C5
the equations described by Wellburn (1994). and C9 tolerated a higher content of NaCl than C7 and C8 (Fig. 1).
Optimal NaCl concentration for C5 and C9 was 11.7 g/L NaCl. Due to
their high tolerances, C5 and C9 were selected for further studies.
2.7. Determination of Na+ , K+ , Ca2+ and Mg2+ in plant tissues In addition, bacterial growth kinetics for C5 and C9 were evaluated
at 0, 2.93, and 5.85 g NaCl/L. The results revealed that NaCl did not
Roots and shoots were washed several times with deionized exert a negative effect on bacterial growth (Figs. 1 and 2).
water. These were oven-dried at 60 ◦ C for 48 h and afterward
ground. Samples of 200 mg of each plant tissue were digested at 3.2. Molecular identification of the C5 and C9 strains
150 ◦ C for 2.0 h in a microwave digester in a mixture of 30% H2 O2 ,
65% HNO3 (Merck, USA), and deionized water in a ratio of 1:1:1 Strains C5, C7, C8, and C9 were presumed to be Azotobacter
to final volume of 12.0 mL; after digestion, volume was adjusted to sp. based on their biochemical and morphological characteristics
20 mL. An Absorption Atomic Spectrophotometer (AAS 2380, Perkin (Table 2). We selected C5 and C9 for further tests due to their ability
Elmer, USA) was employed for measuring the concentration of Na+ , to tolerate high NaCl concentrations. Partial sequence of 16S rDNA
K+ , Ca2+ , and Mg2+ . Reagent blank and analytical duplicates were of C5 and C9 showed 99% and 98% identity with the sequence of
used where appropriate to ensure accuracy and precision of the Azotobacter chroococcum strain IAM 12366, with fragment lengths
analysis. of 1464 and 1377 base pairs, respectively. The partial 16S rDNA
 D. Rojas-Tapias et al. / Applied Soil Ecology 61 (2012) 264–272 267

Fig. 1. Effect of NaCl on bacterial growth. Each value is the mean of three replicates. Error bars represent ±standard deviation.

Table 2
Morphological and biochemical description of strains.

Characteristics C5 C7 C8 C9

Morphological
Gram reaction N N N N
Cell shape SR SR SR SR
Brown pigmentation + + + +
Cyst formation + + + +
Biochemical reactions
Aerobic nitrogen fixation + + + +
Nitrate reduction to nitrite + + + +
Catalase + + + +
Hydrolysis
Urea − − − −
Esculine + + + +
Gelatine − + + +
Carbohydrate utilization
Glucose + + + +
Arabinose − + + −
Mannose + + + +
Mannitol + + + −
N-acetyl-glucosamine − − + +
Maltose + − + +
Caprate − − − −
Malate − + + +
Citrate − − + +
Phenyl-acetate − − + −

+ Indicates a positive reaction while − indicates a negative reaction. N: negative

Gram reaction; P: positive Gram reaction. SR: short rods.

sequences for these strains have been deposited with the EMBL-
EBI/GenBank accession numbers JN683378 and JN683377 for the
A. chroococcum C5 and C9 strains, respectively.

3.3. Plant growth-promoting features

Plant growth-promoting capabilities of the selected strains were

Fig. 2. Effect of NaCl on growth kinetics of Azotobacter sp. (A) C5 and (B) C9. Each
studied (nitrogen fixation, phosphate solubilization, and indole
value is the mean of three replicates. Error bars represent ±standard deviation.
synthesis) at 0, 2.93, and 5.85 g NaCl/L. A. chroococcum C5 and C9
were able to reduce acetylene in both the presence and absence
268 D. Rojas-Tapias et al. / Applied Soil Ecology 61 (2012) 264–272

Table 3
PGP features of strains.

Strain NaCl (g/L) ARA (nmol ethylene/mL h) Indole production (␮g indole/mL) Phosphate solubilization (␮g PO4 3− /mL)

C5 0 138.65 ± 9.77 a 0a 161.11 ± 13.43 a

2.93 130.18 ± 3.50 a 16.06 ± 1.07 b 140.12 ± 26.37 a
5.85 132. 24 ± 1.96 a 16.01 ± 2.46 b 144.74 ± 5.26 a

C9 0 133.84 ± 3.18 a 0a 151.32 ± 7.46 a

2.93 139.33 ± 5.93 a 0a 153.81 ± 17.89 a
5.85 143.22 ± 6.49 a 0a 179.83 ± 2.63 b

± shows standard deviation. Different letters represent significant statistical differences based on Tukey HSD test.

Table 4 bacterial inoculation by 23% and 42% at 2.93 g NaCl/kg soil, and 44%
Chlorophyll content.
and 40% at 5.85 g NaCl/kg soil, for the strains C5 and C9, respec-
Treatment Chlorophyll (mg mL−1 g−1 FW) tively (Fig. 3D). Both bacteria were able to increase the chlorophyll
content at 0 g NaCl/kg soil (Table 4). At 2.93 g NaCl/kg soil Azotobac-
0 g NaCl/kg soil 2.93 g NaCl/kg soil 5.85 g NaCl/kg soil
ter sp. C9 increased chlorophyll content by 30%, while C5 did not
Non-inoculated 2.338 ± 0.023 a 4.063 ± 0.099 b 0.518 ± 0.019 a
exhibit any effect (P > 0.05). And at 5.85 g NaCl/kg soil, both strains
C5 3.780 ± 0.085 b 3.359 ± 0.277 a 2.515 ± 0.111 b
C9 3.560 ± 0.313 b 5.292 ± 0.283 c 3.251 ± 0.281 c increased chlorophyll content by 4- and 6-fold with respect to the
non-inoculated treatment (Table 4).
± shows standard deviation. Different letters represent significant statistical differ-
ences based on Tukey HSD test.
3.5. Effect of bacterial inoculation on Na+ , K+ , Ca2+ and Mg2+
uptake by maize
of NaCl; however, we observed no statistical differences (P > 0.05).
Nitrogen-fixation capacity of C5 and C9 was confirmed by nested
Increasing the NaCl concentration of soil resulted in increased
PCR of nifH genes, which yielded a 370-pb DNA fragment by the suc-
Na+ content in plants (Table 5). In roots, inoculation with bacteria
cessive use of primers nifH(forA) and nifH(rev), and after nifH(forB)
caused a decrease of Na+ content. Similarly, Na+ content in shoots
and nifH(rev). Phosphate solubilization activity was exhibited by
was decreased with inoculation of C5 and C9 at 2.93 g NaCl/kg soil,
both strains C5 and C9; we only observed differences at 5.85 g
and of C9 at 5.85 g NaCl/kg soil. In contrast, the Na+ content was
NaCl/L with strain C9 (P < 0.05). A. chroococcum C9 did not syn-
unaltered by inoculation of bacteria in the non-saline soil.
thesize auxins under the conditions tested, while C5 synthesized
K+ uptake by roots and shoots was increased by bacterial
indole acetic acid but only under saline stress (Table 3).
inoculation, demonstrating a clear bacterial effect on potassium
transport in plants. Accumulation of Na+ and K+ was negatively
3.4. Influence of PGPB and NaCl on maize growth and content of
and positively correlated with plant biomass, respectively (Fig. 4).
photosynthetic pigments
In most cases bacterial inoculation revealed no effects on Ca2+
uptake in roots, while the amount of Ca2+ in shoots was increased.
We tested the influence of PGPB inoculation on maize growth
Finally, the amount of Mg2+ in roots and shoots was diminished
under conditions of slight and moderate salinity. The electrical con-
and increased by inoculation with C5 and C9 under saline stress,
ductivities were 3.054 and 6.004 dS/m for 2.93 and 5.85 g NaCl/kg
respectively (Table 5).
soil, respectively. Parameters such as shoot and root length, and
shoot and root dry weight were evaluated. Inoculation with strains
C5 and C9 increased plant growth, but only under saline stress 3.6. Polyphenol content in leaves
(P < 0.05) (Fig. 3). With 2.93 g NaCl/kg soil, the increases for shoot
length were 45% and 58%, while at 5.85 g NaCl/kg soil the increases Changes in polyphenol content in maize plants were shown
were 27% and 47% for C5 and C9, respectively (Fig. 3A). Similarly, as response to both NaCl and bacterial inoculation (Table 6). The
promotion of shoot dry weight under salt stress was 100% and 95% amount of total polyphenols was enhanced with increasing concen-
at 2.93 g NaCl/kg soil, and 81% and 122% at 5.85 g NaCl/kg soil with trations of salt. Similarly, when bacteria were present, an increase
strains C5 and C9, respectively (Fig. 3C). Increases in root length in content of polyphenols was also observed regardless of salt con-
by bacteria were only exhibited when the concentration of salt centration (Table 6). Azotobacter sp. C5 caused the greatest effect
was 5.85 g NaCl/kg soil (Fig. 3B). Root dry weight was increased by on content of polyphenols in maize leaves (Table 6).

Table 5
PGPB and salt influence on Na+ , K+ , Ca2+ and Mg2+ uptake.

NaCl (g/kg soil) Treatment Root (mg/g DW) Shoot (mg/g DW)

Na+ K+ Ca2+ Mg2+ Na+ K+ Ca2+ Mg2+

0 Non-inoc. 2.86 a 20.51 f 2.38 a 1.47 a 0.20 a 51.24 c 3.67 a 2.51 a

C5 2.70 a 18.55 e 2.05 a 1.54 a 0.41 a 50.57 c 2.60 a 2.37 a
C9 2.61 a 18.34 e 2.13 a 1.43 a 0.40 a 61.21 d 3.57 a 2.20 a

2.93 Non-inoc. 16.09 c 8.19 c 2.35 a 2.42 b 17.97 c 40.42 b 3.48 a 2.97 b
C5 15.59 b 8.53 c 2.25 a 1.61 a 16.02 b 37.60 b 3.23 a 3.25 c
C9 14.08 b 11.41 d 1.83 a 1.76 a 16.32 b 42.35 b 3.96 b 3.04 b

5.85 Non-inoc. 18.93 d 3.53 a 3.92 b 3.12 c 44.88 e 25.20 a 4.65 b 3.15 b
C5 21.77 e 4.90 b 2.53 a 2.40 b 43.37 e 34.67 b 5.47 c 5.61 d
C9 15.18 b 8.41 c 2.57 a 2.38 b 32.56 d 36.68 b 5.65 c 3.45 c

Different letters represent significant statistically differences based on Tukey HSD test.
 D. Rojas-Tapias et al. / Applied Soil Ecology 61 (2012) 264–272 269

Fig. 3. Effect of NaCl and inoculation with C5 and C9 on plant biomass expressed as: (A) shoot length, (B) root length, (C) shoot dry weight, and (D) root dry weight. Each
value is the mean of six replicates. Error bars represent ±standard deviation. Different letters represent significant statistical differences based on Tukey HSD test (P < 0.05).

Table 6 bacterial inoculation. Proline content in bacterial-inoculated plants

Measuring of total polyphenols in leaves.
was enhanced in the non-saline soil by 76 and 84% for C5 and C9,
Treatment Total polyphenols (mg/g FW) respectively.
0 g NaCl/kg soil 2.93 g NaCl/kg soil 5.85 g NaCl/kg soil

Non-inoculated 2.858 ± 0.363 a 3.210 ± 0.340 a 3.834 ± 0.360 a 4. Discussion

C5 3.468 ± 0.133 b 4.110 ± 0.036 b 4.937 ± 0.446 b
C9 3.612 ± 0.298 b 3.531 ± 0.256 a 4.902 ± 0.103 b Salinity affects plant growth by imposing both ionic and osmotic
± shows standard deviation. Different letters represent significant statistical differ- stresses (Shabala and Cuin, 2008). We observed that regardless
ences based on Tukey HSD test. of biological treatment salinity negatively affected plant growth
(length and weight). Because of the osmotic gradient generated,
elevated Na+ levels in the soil solution drive water out of the cell
3.7. Proline content in leaves reducing almost instantaneously cell turgor, leaf area, and con-
sequently the photosynthetic activity and carbon fixation (Yeo
Our findings indicated that under saline stress plants synthe- et al., 1991). In the current study, A. chroococcum-inoculated plants
sized proline to a greater extent (Table 7). However, the content had significantly higher biomass than their respective controls in
of proline with both slight and moderate salinity was decreased by the saline soil; however, no effects were observed under non-
restrictive growth conditions. Moreover, the effect of salinity on
Table 7
synthesis of photosynthetic pigments depended on the specific
Proline in leaves. concentration of NaCl. Nevertheless, the inoculation with C5 and C9
enhanced the content of chlorophyll revealing a positive effect on
Treatment Proline (␮mol/g FW)
growth and plant development. Early studies have shown that syn-
0 g NaCl/kg soil 2.93 g NaCl/kg soil 5.85 g NaCl/kg soil thesis and activity of photosynthetic pigments could be repressed
Non-inoculated 3.156 ± 0.058 a 5.781 ± 0.580 b 6.283 ± 0.552 c by excessive concentrations of Na+ (Parida et al., 2004); However,
C5 5.569 ± 0.219 b 3.995 ± 0.087 a 4.797 ± 0.261 a studies reported by Hamdia et al. (2004) showed that inocula-
C9 5.800 ± 0.472 b 5.355 ± 0.291 b 5.443 ± 0.376 b tion with Azospirillum lipoferum, also a PGPB, ZA/I improved plant
± shows standard deviation. Different letters represent significant statistical differ- dry weight and leaf area in maize under high salinity. Similarly,
ences based on Tukey HSD test. Mayak et al. (2004) observed that tomato plants inoculated with
270 D. Rojas-Tapias et al. / Applied Soil Ecology 61 (2012) 264–272

Fig. 4. Correlations of plant biomass (root weight + shoot weight) with Na+ in (A) roots and (B) shoots, and K+ content in (C) roots and (D) shoots. *, **, *** significant at
P < 0.05, 0.01 and 0.001, respectively. At equations, C5, C9, and NI indicate inoculation with A. chroococcum C5 and C9, and non-inoculated treatment, respectively.

Achromobacter piechaudii ARV8 had increased plant biomass under improving the K+ /Na+ ratio. In this case, A. chroococcum C9 exhib-
120 and 207 mM NaCl. These results indicated that inoculation with ited the greatest effect. In addition, accumulation of Ca2+ and Mg2+
the selected bacterium could decrease the injurious effects caused in roots decreased as the concentration of Na+ in soil increased.
by salinity. Likely, Na+ exerted ionic competence in soil, diminishing the abil-
High salinity can also affect the growth and role of bacteria in ity for ion uptake by the plant. In shoots, however, an increase
the environment (Steinborn and Roughley, 1974). However, we in the content of Ca2+ and Mg2+ mediated by bacterial inocula-
observed that growth and PGP features of strains C5 and C9 were tion was observed, which may be explained by an increase in
not negatively influenced by salinity. Bacteria can synthesize com- mineral availability mediated by the bacterial metabolism (e.g.
patible solutes (sugars, amino acids, or derivatives) that act as releasing of organic acids). Similar results were reported by Ashraf
osmolytes and help organisms to survive when there is extreme et al. (2004), who found that inoculation with exopolysaccharide-
osmotic stress (Bacilio et al., 2004; da Costa et al., 1998; Parida and producing bacteria could restrict Na+ influx into roots. Further,
Das, 2005). Bacterial traits, such as nitrogen fixation, phosphate Zhang et al. (2008) reported that inoculation with Bacillus sub-
solubilization, and IAA synthesis, have exhibited an influence on tilis GB03 could mediate the level of salt tolerance in Arabidopsis
plant growth by increasing nutrient availability and by influencing thaliana through regulation of the potassium transporter HKT1.
plant development (Glick, 2010). Therefore, growth promotion by These results supported the idea that bacteria can mediate the
strains C5 and C9 may be mediated by these traits. A. chroococcum expression of an ion high-affinity K+ transporter in Arabidopsis.
C5 and C9 were able to solubilize tricalcium phosphate and to fix Salinity decreases carbon uptake by limiting photosynthesis,
nitrogen, but only C5 was able to synthesize IAA, indicating plant causing an over-reduction of photosynthetic electron chain, and
growth-promoting characteristics. redirecting the photon energy into processes that favor the produc-
Na+ exclusion and K+ influx are the most important plant strate- tion of Reactive Oxygen Species (ROS) (Hichem et al., 2009; Johnson
gies for alleviating salt-induced stress (Fortmeier and Schubert, et al., 2003). Synthesis of polyphenols by plants constitutes one of
1995; Shabala and Cuin, 2008). We observed that high levels of Na+ the adaptive mechanisms for reducing oxidative damage (Hichem
in roots and shoots were negatively correlated with maize biomass, et al., 2009; Nautiyal et al., 2008). In this study, salinity signifi-
regardless of bacterial treatment employed. Conversely, K+ con- cantly increased polyphenols content in maize leaves and bacterial
tent in plants was correlated positively with plant biomass. The inoculation also improved the amount of polyphenols compared
present results showed that salinity increased Na+ and decreased with the respective non-inoculated control. Polyphenols can elim-
K+ concentration, thus decreasing the K+ /Na+ ratio with increas- inate radical species, thus preventing the propagation of oxidative
ing salinity stress. However, bacterial inoculation resulted in chain reactions (Rice-Evans et al., 1997). Nautiyal et al. (2008) sim-
significantly decreased Na+ and increased K+ concentration, ilarly showed that inoculation with PGPB Bacillus lentimorbus NRRL
 D. Rojas-Tapias et al. / Applied Soil Ecology 61 (2012) 264–272 271

B-30488 could mediate induction of dietary antioxidant in veg- Costacurta, A., Vanderleyden, J., 1995. Synthesis of phytohormones by plant-
etables and fruit expressed as total polyphenol content. However, associated bacteria. Crit. Rev. Microbiol. 21, 1–18.
da Costa, M., Santos, H., Galinski, E., 1998. An overview of the role and diversity
few data are available about the mechanisms involved in bacterial- of compatible solutes in Bacteria and Archaea. Biochem. Eng. Biotechnol. 61,
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Studies carried out by Hamdia et al. (2004) and Nadeem et al. Dimkpa, C., Weinand, T., Asch, F., 2009. Plant–rhizobacteria interactions alleviate
abiotic stress conditions. Plant Cell Environ. 32, 1682–1694.
(2007) showed that plant proline contents are increased by saline Eckert, B., Weber, O.B., Kirchhof, G., Halbritter, A., Stoffels, M., Hartmann, A., 2001.
stress, but decreased by inoculation with PGPB. In this study, we Azospirillum doebereinerae sp. nov. a nitrogen-fixing bacterium associated with
also found that proline content in leaves increased with increas- the C4-grass Miscanthus. Int. J. Syst. Evol. Microbiol. 51, 17–26.
Fiske, C.H., Subbarow, Y., 1925. The colorimetric determination of phosphorus. J.
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C9 significantly decreased the proline concentration in leaves. To Fortmeier, R., Schubert, S., 1995. Salt tolerance of maize (Zea mays L.): the role of
date, there is evidence indicating a positive correlation among pro- sodium exclusion. Plant Cell Environ. 18, 1041–1047.
Fu, Q., Liu, C., Ding, N., Lin, Y., Guo, B., 2010. Ameliorative effects of inoculation with
line accumulation and adaptation to salt stress, but the results are
the plant growth-promoting rhizobacterium Pseudomonas sp. DW1 on growth
still controversial (Ashraf and Foolad, 2007; Chandler and Thorpe, of eggplant (Solanum melongena L.) seedlings under salt stress. Agric. Water
1987). Our observation revealed that under salt-mediated stress, Manage. 97, 1994–2000.
inoculation with C5 and C9 decreased proline concentration con- Gauri, S.S., Mandal, S.M., Mondal, K.C., Dey, S., Pati, B.R., 2009. Enhanced produc-
tion and partial characterization of an extracellular polysaccharide from newly
comitantly with increased plant biomass. Notably, in the absence isolated Azotobacter sp. SSB81. Biores. Technol. 100, 4240–4243.
of salinity, bacteria significantly increased the proline content in Glick, B.R., 2010. Using soil bacteria to facilitate phytoremediation. Biotechnol. Adv.
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Glickmann, E., Dessaux, Y., 1995. A critical examination of the specificity of the
plant growth. This information may support the results obtained in salkowski reagent for indolic compounds produced by phytopathogenic bacte-
pot experiments where no effects on plant biomass were observed ria. Appl. Environ. Microbiol. 61, 793–796.
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research and extension needs. Field Crop Res. 65, 93–106.
the strains EZB4 of Arthrobacter sp. and EZB8 of Bacillus sp. increased Hamdia, M.A.E.-S., Shaddad, M.A.K., Doaa, M.M., 2004. Mechanisms of salt toler-
the content of proline even in the absence of abiotic stress. They ance and interactive effects of Azospirillum brasilense inoculation on maize
argue that bacteria likely exerted some biotic stress that triggered cultivars grown under salt stress conditions. Plant Growth Regul. 44, 165–
174.
proline biosynthesis in plants. Hichem, H., Mounir, D., Naceur, E.A., 2009. Differential responses of two maize (Zea
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5. Conclusions and oxidative damages. Ind. Crop Prod. 30, 144–151.
Hiscox, J.D., Israelstam, G.F., 1979. A method for the extraction of chlorophyll from
leaf tissue without maceration. Can. J. Bot. 57, 1332–1334.
We demonstrated that inoculation with A. chroococcum strains Johnson, S.M., Doherty, S.J., Croy, R.R.D., 2003. Biphasic superoxide generation
C5 and C9 protected plants against the inhibitory effects of NaCl. in potato tubers. A self-amplifying response to stress. Plant Physiol. 131,
1440–1449.
We argue on the basis of our findings that bacterial amelioration of
Mayak, S., Tirosh, T., Glick, B.R., 2004. Plant growth-promoting bacteria confer
saline stress could be the integration of several aspects including resistance in tomato plants to salt stress. Plant Physiol. Biochem. 42, 565–
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Munns, R., Tester, M., 2008. Mechanisms of salinity tolerance. Annu. Rev. Plant Biol.
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an improved K+ /Na+ ratio in plants. However, extensive research tions on inducing salt tolerance in maize through inoculation with rhizobacteria
is needed to elucidate how bacteria mediate this effect on maize containing ACC deaminase activity. Can. J. Microbiol. 53, 1141–1149.
Nautiyal, C.S., Govindarajan, R., Lavania, M., Pushpangadan, P., 2008. Novel mech-
growth. In summary, results indicated that bacteria could amelio- anism of modulating natural antioxidants in functional foods: involvement of
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Parida, A., Das, A.B., Mittra, B., 2004. Effects of salt on growth, ion accumulation,
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