Post on 16-Mar-2023
ORIGINAL PAPER
Cyanobacteria-mediated phenylpropanoidsand phytohormones in rice (Oryza sativa) enhance plantgrowth and stress tolerance
Dhananjaya P. Singh • Ratna Prabha •
Mahesh S. Yandigeri • Dilip K. Arora
Received: 29 March 2011 / Accepted: 11 June 2011 / Published online: 6 July 2011
� Springer Science+Business Media B.V. 2011
Abstract Phenylpropanoids, flavonoids and plant
growth regulators in rice (Oryza sativa) variety (UPR
1823) inoculated with different cyanobacterial strains
namely Anabaena oryzae, Anabaena doliolum, Pho-
rmidium fragile, Calothrix geitonos, Hapalosiphon
intricatus, Aulosira fertilissima, Tolypothrix tenuis,
Oscillatoria acuta and Plectonema boryanum were
quantified using HPLC in pot conditions after 15 and
30 days. Qualitative analysis of the induced com-
pounds using reverse phase HPLC and further
confirmation with LC-MS/MS showed consistent
accumulation of phenolic acids (gallic, gentisic,
caffeic, chlorogenic and ferulic acids), flavonoids
(rutin and quercetin) and phytohormones (indole
acetic acid and indole butyric acid) in rice leaves.
Plant growth promotion (shoot, root length and
biomass) was positively correlated with total protein
and chlorophyll content of leaves. Enzyme activity of
peroxidase and phenylalanine ammonia lyase and
total phenolic content was fairly high in rice leaves
inoculated with O. acuta and P. boryanum after
30 days. Differential systemic accumulation of phe-
nylpropanoids in plant leaves led us to conclude that
cyanobacterial inoculation correlates positively with
plant growth promotion and stress tolerance in rice.
Furthermore, the study helped in deciphering possible
mechanisms underlying plant growth promotion and
stress tolerance in rice following cyanobacterial
inoculation and indicated the less explored avenue
of cyanobacterial colonization in stress tolerance
against abiotic stress.
Keywords Cyanobacteria � PGPR � Rice �Phenolics � Flavonoids � Phytohormones
Introduction
Cyanobacteria (blue-green algae) are prominent
inhabitants of many agricultural soils and are the
most natural colonizers of rice roots (Khan et al.
1994; Vaishampayan et al. 2001) where they poten-
tially contribute towards biological nitrogen fixation
(Rai et al. 2000), phosphate solubilization (Yandigeri
et al. 2010) and mineral release to improve soil
fertility and crop productivity (Fernandez et al.
2000). Besides naturally fertilizing and balancing
mineral nutrition in the soils, many organisms are
known to produce growth promoting substances that
enhance plant health by a plethora of mechanisms
(Karthikeyan et al. 2007).
Non-pathogenic plant growth-promoting rhizo-
bacteria (PGPRs) (Kloepper et al. 1980) play critical
role in plant health and nutrition (Ahmad et al.
2008). They can benefit plant growth by improving
D. P. Singh (&) � R. Prabha � M. S. Yandigeri �D. K. Arora
National Bureau of Agriculturally Important
Microorganisms, Kushmaur, Maunath Bhanjan 275101,
India
e-mail: dpsfarm@rediffmail.com
123
Antonie van Leeuwenhoek (2011) 100:557–568
DOI 10.1007/s10482-011-9611-0
plant nutrition and soil fertility (Glick 1995;
Bloemberg and Lugtenberg 2001), producing plant
growth regulators (Gutierrez et al. 2001), evoking
changes in metabolic status of plants (Pieterse et al.
1996a,b; M’Piga et al. 1997; Sarma et al. 2002;
Singh et al. 2002, 2003; Yedidia et al. 2003),
inducing systemic resistance against pathogenic
attack (Ahmad et al. 2008; Moon et al. 2008;
Rodriguez-Diaz et al. 2008; Barriuso et al. 2008),
rhizoremediation and plant stress control (Lugten-
berg and Kamilova 2009). It is widely realized that
in plants, the physiological disorders due to abiotic
stresses or pathological disorders caused by micro-
bial agents that promote the development of hyper-
sensitive reactions, usually involve destructive free
radical-mediated oxidative degradation of biomole-
cules (Senaratna et al. 2000). Studies suggest that
plants defend themselves from such oxidative dam-
ages by the changes in their physiological and
biochemical status (Senaratna et al. 1987) and root
colonization with PGPRs facilitate plants to fight
against pathogen or abiotic stress mediated losses
(Barriuso et al. 2008; Lugtenberg and Kamilova
2009). Rhizobacteria-elicited ‘‘induced systemic tol-
erance’’ (IST) that can enhance tolerance in plants
to abiotic stress due to physical and chemical
changes (Yang et al. 2008) is a relatively recent
approach that considerably overlaps with the mech-
anisms of systemic induced resistance in plants
(Ramos Solano et al. 2008). Since cyanobacteria
naturally colonize rice roots in salt affected soils, it
is imperative to suggest their role as PGPR and
therefore, it is hypothesized that their colonization
helps plants to promote growth in stressed soil
conditions because of the elicitation of induced
systemic responses in plant leaves. Release of a
diverse array of biologically active metabolites by
the growing cyanobacterial cells (Kumar et al. 2000;
Cryl and Karl 2008; Wink and Schimmer 1999;
Dixon 2001; Rastogi and Sinha 2009) in the
rhizosphere soil may also assist in enhancing plant
growth in salt stressed soils.
In present communication, we report impact of
cyanobacterial colonization on the physical growth,
metabolic (phenylpropanoids and phytohormones)
and enzymatic (peroxidase and phenylalanine ammo-
nia lyase; PAL) status of rice in planta under stressed
soil and in the root rhizosphere.
Materials and methods
The microorganisms, growth conditions and seed
treatment
Cyanobacterial strains namely Anabaena oryzae,
A. doliolum, Phormidium fragile, Calothrix geitonos,
Hapalosiphon intricatus, Aulosira fertilissima,
Tolypothrix tenuis, Oscillatoria acuta and Plecto-
nema boryanum were obtained from the NAIMCC
culture collection, Maunath Bhanjan, India. All the
strains were transferred from their respective slants in
40 ml (93) of BG11 medium (Stanier et al. 1971) in
100 ml flasks. Cultures were bubbled with air con-
taining 1% (v/v) carbon dioxide and were kept under
continuous illumination at 70 l Em-2 s-2 from
incandescent lamps with 12 h light–dark cycles at
25 ± 2�C. Cells (120 ml) were harvested in the mid
to late-exponential phase of growth by centrifugation
(50009g) at room temperature and cell pellet
(200 mg) was finally suspended in 1 ml double
distilled water (DDW) containing 1.0% carboxy-
methylcellulose (CMC) as binder.
Rice seeds (variety UPR 1823) were obtained from
the Directorate of Seed Research, Maunath Bhanjan,
India. Surface sterilized seeds were pre-soaked in
DDW and kept on sterilized moist Whatman filter
paper one day before the inoculation was made.
Thirty seeds were transferred to glass tubes contain-
ing 5.0 ml cyanobacterial cell suspension
(OD663nm * 0.67) and left for 4 h. Inoculated seeds
were sown in sterilized plastic pots (6 seeds per pot of
15 cm dia.) filled with sterile soil (pH 8.8, EC 5.2 dS/
m) containing sand (3:1, w/w) and pots were
transferred to the culture room maintained with
fluorescent light at 25 ± 2�C. Plants were allowed
to grow in four replications (6 plants per pot, 24
plants per treatment) and harvested at 15 and 30 days.
Plants maintained in similar growth conditions but
remained uninoculated served as control.
Plant growth assessment
Ten rice plants per treatment and control were
randomly harvested from the pots after 15 and
30 days of inoculation. Root and shoot length and
fresh weight of each and every plant were measured
immediately after harvesting.
558 Antonie van Leeuwenhoek (2011) 100:557–568
123
Biochemical tests
Chlorophyll content in rice leaves was quantified by
extracting the pigment from 0.5 g freshly harvested
leaf tissues using methanol: water (9:1, v/v). After
removal of the precipitate through centrifugation at
150009g for 5 min, chlorophyll in the supernatant
was quantified in terms of A665nm (Ferjani et al.
2003).
Total protein content (TPC) was extracted as per
the method described by Ferjani et al. (2003). One g
of freshly harvested rice leaves was macerated with
1% tricholoroacetic acid (5 ml) and the precipitate
was separated by centrifugation at 150009g for
10 min at 4�C. The pellet was resuspended in 1 N
NaOH (5 ml), boiled for 30 min, cooled and centri-
fuged at 150009g for 5 min. The supernatant was
quantified for total content of protein as per the
method of Lowry et al. (1951) with bovine serum
albumin (BSA) as standard.
Total soluble phenol (TSP) was estimated spectro-
photometrically using the Prussian blue method as
described by Graham (1992) and expressed in terms of
gallic acid equivalents by using gallic acid (HiMedia,
India) as standard. Absorbance was recorded at
700 nm using UV–VIS spectrophotometer (Shimadzu
Corporation, Japan). Analytical grade reagents were
used throughout the experiments.
Enzyme assays
Enzyme extract from fresh rice leaves (1 g) collected
after 15 and 30 days of inoculation was extracted in
3 ml of 0.05 M sodium phosphate buffer (pH 7.8)
containing 1 mM EDTA and 2% (w/v) polyvinylpyr-
rolidone. The supernatant used as enzyme extract was
obtained from the homogenate by centrifugation at
130009g for 15 min at 4�C. Peroxidase activity was
measured using a modified procedure of Egley et al.
(1983). The reaction mixture (total volume 2 ml)
contained 25 mM sodium phosphate buffer (pH 7.0),
0.1 mM EDTA, 0.05% guaiacol (2-ethoxyphenol),
1.0 mM H2O2 and 100 ll enzyme extract. The
increase in the absorbance due to oxidation of guaiacol
was measured at 470 nm (E = 26.6 mM-1cm-1).
PAL activity was estimated from the same enzyme
extract as per the method described by Singh et al.
(2003).
Extraction of phenolics and phytohormones
Phenolics were extracted from the freshly harvested
rice leaves as described earlier (Singh et al. 2003).
Briefly, 1 g leaf tissues was macerated in a pestle-
mortar and then mixed with 5 ml of extraction
solvent methanol: water (1:1, v/v). Samples were
collected in screw-capped tubes and the suspension
was subjected to ultrasonication for 15 min at room
temperature followed by centrifugation at 75009g for
15 min. The clear greenish supernatant was collected
and the cell debris was again suspended in extraction
solvent and kept for 4 h. Finally the supernatants
were pooled and thoroughly mixed with a pinch of
charcoal to remove the pigments. The clear superna-
tant thus obtained was filtered and the solvent was
evaporated under vacuum. Dried samples were re-
dissolved in HPLC grade methanol by vortexing and
stored at 4�C for further analysis.
For the extraction of phytohormones, one g fresh
leaves were homogenized with 80% methanol con-
taining butylated hydroxytoulene (BHT, 100 mg/l)
and the homogenates were kept overnight at 4�C in
dark. After re-extraction (93) with 80% methanol,
the resulting supernatant was frozen at -20�C,
thawed and centrifuged at 90009g for 30 min at
4�C to remove impurities. The resultant was redis-
solved in HPLC grade methanol for the estimation of
phytohormones.
Metabolic profiling of rhizospheric soil containing
root exudates (Walker et al. 2003b) from each
treatment was carried out by drying one g soil under
vacuuo and suspending the same in methanol : water
(1:1, v/v, 5 ml) thrice. The supernatants were pooled
together and solvent was evaporated to dryness. The
dried extract was redissolved in methanol (HPLC
grade) and subjected to filtration prior to analysis.
HPLC analysis
High performance liquid chromatography (HPLC) of
rice leaves and rhizospheric soil extracts was
performed using HPLC system (Waters, USA)
equipped with binary Waters 515 reciprocating
pumps, a variable photodiode array (PDA) detector
(Waters 2996) and system controller equipped with
Waters�EmpowerTM
software for data integration and
analysis. Reverse phase liquid chromatographic
analysis of the samples (injection volume 10 ll)
Antonie van Leeuwenhoek (2011) 100:557–568 559
123
was carried out in isocratic mode on a C-18 column
(250 9 4.6 mm i.d., 5 lm particle size) at 25 ± 1�C
at a flow rate of 1 ml/min of the mobile phase
methanol: 0.4% acetic acid in water (60:40, v/v) and
detection at 254 and 280 nm for phenolic acids
gallic, ferulic, chlorogenic, gentisic and cinnamic
acids. Flavonoids (rutin and quercetin) and phyto-
hormones (indole acetic and indole butyric acid)
were analyzed as per the method of Carreno-Lopez
et al. (2000) at a flow rate of 1 ml/min of methanol:
1% aqueous acetic acid (24:76, v/v) as mobile phase.
Samples were subjected to membrane filtration
through 0.45 lm membrane filter prior to injection
in the sample loop. HPLC grade solvents and
chemicals (E Merck and Hi Media, India) were used
throughout the analysis. Qualitative characterization
of the compounds in the sample was done by
comparing retention time (Rt) and co-injection while
quantitative analysis was performed by comparing
peak areas of the standard compounds obtained from
Hi-Media, India.
Qualitative LC-MS/MS
Phenolic compounds used as reference standards and
their presence in the samples was further validated by
mass spectrometric analysis (Triple-quadrupole mass
spectrometer, API 2000, Applied Biosystems,
Ontario, Canada) as per the methods described earlier
(Singh et al. 2009; Niranjan et al. 2009). The
compounds were detected according to their respec-
tive m/z values of their parent and product ions; gallic
acid (169/125), caffeic acid (179/135), chlorogenic
acid (353/191), ferulic acid (193/134) and quercetin
(301/151).
Statistical analysis
The data were subjected to t-test and analysis of
variance (ANOVA) in Duncan’s multiple range test
with the software SPSS for windows 8.0. Differences
were considered to be significant at the 95% confi-
dence level. Results were reported as mean (±)
standard deviation (SD) of four replicates from pot
experiments and three from sample analysis using
HPLC.
Results
Rice plants inoculated with different strains of
cyanobacteria showed differential responses in terms
of shoot and root length and plant weight (Figs. 1, 2).
Maximum shoot and root length and plant weight
(wt) (17.6 cm, 7.3 cm and 2.18 g after 15 days and
24.1 cm, 8.6 cm and 3.1 g after 30 days, respec-
tively) was observed in the plants inoculated with
P. boryanum. Quantitative profile of different bio-
chemicals viz., chlorophyll and total protein in rice
leaves varied significantly within the plants inocu-
lated with different cyanobacterial strains (Fig. 3).
Chlorophyll content in plant leaves ranged from
57.43 mg/g in A. oryzae to 143.2 mg/g fresh wt in
P. boryanum and total protein 9.34 mg/g in P. fragile
to 17.86 mg/g fresh wt in T. tenuis after 30 days of
inoculation (Fig. 3). In comparison to the uninocu-
lated (control) plants, all the treatments performed
significantly well in terms of the physical and
biochemical growth indicators (Figs. 1, 2, 3). Growth
promotion effect as well as the colonization of soil
and plant root by a potential cyanobacterial strain is
also shown in Fig. 4a and b.
Consistent systemic accumulation of phenolic acids
(gallic, caffeic, chlorogenic and ferulic acids) was
observed in the leaves of inoculated plants after 15 and
30 days of growth (Table 1). In certain treatments the
presence of chlorogenic acid in A. fertilissima inocu-
lated plant leaves and ferulic acid in H. intricatus
inoculated plant leaves could not be traced after
Fig. 1 Effect of cyanobacterial inoculation on shoot and root
length of rice, strains—1. Anabaena oryzae, 2. A. doliolum, 3.
Phormidium fragile, 4. Calothrix geitonos, 5. Hapalosiphonintricatus, 6. Aulosira fertilissima, 7. Tolypothrix tenuis, 8.
Oscillatoria acuta, 9. Plectonema boryanum, 10. Control; two
population t-test: shoot length t = 5.77, P = 1.79978E-5; root
length t = 2.59, P = 0.0183; at 0.05 level, the two means are
significantly different
560 Antonie van Leeuwenhoek (2011) 100:557–568
123
15 days of inoculation, although the compounds
appeared in the leaves after 30 days. After 15 days,
maximum accumulation of gallic, caffeic, chlorogenic
and ferulic acids (111.4, 5.62, 3.60 and 9.97 lg/g fresh
wt) was recorded in rice leaves inoculated with
H. intricatus, A. doliolum, A. oryzae and O. acuta
respectively. However, after 30 days of inoculation,
the in planta accumulation was 144.7, 8.27, 6.50
and 3.63 lg/g fresh wt respectively in O. acuta,
P. boryanum, A. oryzae and A. doliolum inoculated
plant leaves.
Rice leaves showed maximum accumulation of
total phenol following inoculation with A. oryzae
(Fig. 5). It is evident that 30 days after inoculation
favored total phenol content in rice leaves. Results
on peroxidase (Fig. 6) and PAL activity (Fig. 7) in
the leaves of the plants inoculated with cyanobac-
terial strains showed enhanced enzyme activity after
15 and 30 days. Almost similar trend was observed
with both the enzymes. Plants inoculated with
O. acuta and P. boryanum showed high peroxi-
dase and PAL activity while all other treatments
induced enzyme activity as compared to control.
0 1 2 3 4 5 6 7 8 9 101.0
1.5
2.0
2.5
3.0
Pla
nt fr
esh
wt (
g)
Cyanobacterial strains
After 15 days of inoculation After 30 days of inoculation
Fig. 2 Effect of cyanobacteria inoculation on fresh weight of
rice plants, strains—1. Anabaena oryzae, 2. A. doliolum, 3.
Phormidium fragile, 4. Calothrix geitonos, 5. Hapalosiphonintricatus, 6. Aulosira fertilissima, 7. Tolypothrix tenuis, 8.
Oscillatoria acuta, 9. Plectonema boryanum, 10. Control; two
population t-test: Plant wt t = 3.85, P = 0.00117, at 0.05 level,
the two means are significantly different
0 1 2 3 4 5 6 7 8 9 100
20
40
60
80
100
120
140
160
180
200
Qua
ntity
(m
g/g
fres
h w
t)
cyanobacterial strains
total protein after 15 days total protein after 30 days Chl content after 15 days Chl content after 30 days
Fig. 3 Quantitative profile of different biochemicals (total
protein and chlorophyll) in leaves of rice plants inoculated
with different cyanobacterial strains— 1. Anabaena oryzae,
2. A. doliolum, 3. Phormidium fragile, 4. Calothrix geitonos,
5. Hapalosiphon intricatus, 6. Aulosira fertilissima, 7. Tolypo-thrix tenuis, 8. Oscillatoria acuta, 9. Plectonema boryanum,
10. Control. Total protein in terms of bovine serum albumin;
two population t-test for total protein t = 6.87111, P=
1.99046E-6; Chlorophyll content: t = 3.14677, P = 0.00558,
at 0.05 level, the two means are significantly different
Fig. 4 a Colonization of inoculated cyanobacterium Oscilla-toria acuta on the soil surface; and b root colonization with
O. acuta
Antonie van Leeuwenhoek (2011) 100:557–568 561
123
Phenolic acids (gallic, gentisic, chlorogenic and
ferulic acids) and flavonoids (rutin and quercetin) in
the rhizosphere soil was consistently observed at
15 and 30 DAI and gallic acid was found to
be maximum in all the treatments (Table 2).
P. boryanum inoculation showed maximum gallic
acid content (170.13 lg/g) after 30 DAI in the
rhizosphere soil followed by gentisic acid (9.47 lg/
g). Similarly, maximum content of chlorogenic
acid (7.26 lg/g) was found in rhizosphere soil of
Table 1 Accumulation of phenolic acids in leaves of rice plants after 15 and 30 days of cyanobacterial inoculation
Treatments Phenolic acids (lg/g fresh wt)
15 DAI 30 DAI
Gallic Caffeic Chlorogenic Ferulic Gallic Caffeic Chlorogenic Ferulic
Anabaena oryzae 75.00 c 1.30 de 3.60 a 2.50 c 84.43 f 0.76 f 6.50 a 1.67 c
Anabaena doliolum 34.63 f 5.62 a 1.70 d 3.63 b 52.17 h 4.63 c 3.30 d 3.63 a
Phormidium fragile 28.17 g 2.60 cd 2.50 b 1.90 de 45.03 i 2.47 e 1.50 ef 2.70 b
Calothrix geitonos 61.17 d 1.37 de 2.70 b 1.80 de 109.73 d 0.80 f 0.93 fg 0.83 d
Hapalosiphon intricatus 111.4 a 1.62 de 1.73 d 0.70 g 88.07 e 6.40 b 1.63 e 0.83 d
Aulosira fertilissima 75.83 c 3.57 bc 0.98 e 2.37 cd 129.50 c 2.37 e 1.37 efg 2.60 b
Tolypothrix tenuis 56.27 e 1.16 e 2.10 c 1.40 ef 67.00 g 2.57 e 1.21 efg 1.13 d
Oscillatoria acuta 90.43 b 4.16 b 2.63 b 9.97 a 144.70 a 3.40 d 4.23 c 3.80 a
Plectonema boryanum 87.57 b 2.13 de 2.53 b 1.00 fg 135.23 b 8.27 a 5.60 b 1.67 c
Control 20.27 h 1.54 de 0.82 e 0.50 g 22.63 j 1.00 f 0.84 g 0.80 d
SEM± 1.32 0.42 0.100 0.185 0.872 0.184 0.195 0.145
CD (P = 0.05) 3.93 1.23 0.298 0.551 2.59 0.549 0.578 0.431
CV % 3.6 28.6 8.2 12.5 1.7 9.8 12.4 12.8
HPLC retention time (Rt) gallic acid 2.53, caffeic 3.22, chlorogenic 3.56, ferulic 4.32 min
DAI days after inoculation, SEM± standard error of means±, CD critical difference (at significance of 95%), CV coefficient of
variance; Values in the same column followed by a different letter are significantly different (a = 0.05) in Duncan’s multiple range
test
Fig. 5 Total phenol content in terms of gallic acid equivalents
in the leaves of rice plants inoculated with different cyano-
bacterial strains—1. Anabaena oryzae, 2. Anabaena doliolum,
3. Phormidium fragile, 4. Calothrix geitonos, 5. Hapalosiphon
intricatus, 6. Aulosira fertilissima, 7. Tolypothrix tenuis, 8.
Oscillatoria acuta, 9. Plectonema boryanum, 10. Control; One
population t-test for total phenol : t = 2.44946, P = 0.02477,
at 0.05 level, the two means are significantly different
562 Antonie van Leeuwenhoek (2011) 100:557–568
123
A. oryzae, ferulic acid (4.60 lg/g) in O. acuta, rutin
(5.76 lg/g) in A. fertilissima and quercetin (9.68
lg/g) in P. boryanum inoculated plants (Table 2).
Inoculation of cyanobacterial strains favored
enhanced content of compounds in comparison to
control. Qualitative and quantitative analysis of the
compounds was performed by reverse-phase HPLC
and further validated by the LC-MS/MS analysis.
The level of phytohormones (IAA and IBA) was
determined in rice leaves (Fig. 8) and the rhizospher-
ic soil (Fig. 9) inoculated with different cyanobacte-
rial strains. Although the presence of IAA was more
prevalent quantitatively than the IBA, its presence
was directly correlated with the root or shoot length.
Corresponding to the high levels of IAA and IBA in
leaves, plant height showed increasing trend except in
certain treatments (Fig. 1). The level of phytohor-
mones in plant leaves was very high as compared to
control and so was the plant height. Interestingly, the
level of phytohormones in the rhizospheric soil was
also fairly high and corresponded with root length
(Fig. 9). Overall, inoculation of rice plants with
P. boryanum showed high content of phytohormones
that was reflected in terms of root and shoot length.
Discussion
Concomitant qualitative and quantitative alterations
in secondary metabolites in leaves and rhizospheric
soil of plants inoculated with certain cyanobacterial
strains is positively correlated with growth (root,
shoot length, biomass accumulation, chlorophyll, and
protein content) of rice grown under stress soil (pH
8.8, EC 5.2 dS/m). Results indicated systemic
accumulation of phenylpropanoid metabolites,
enhanced content of total phenol, peroxidase and
PAL enzyme and induced accumulation of phytohor-
mones in plant leaves. Simultaneously, increased
levels of phenolics, flavonoids and phytohormones in
the root rhizosphere were also observed.
Reduced growth and development of rice plants
due to salt stress in terms of damaged biochemical
and physiological mechanisms is documented (Fadz-
illa et al. 1997). However, such stresses in plants may
be believed to some extent by the application of
rhizobacterial inoculants which evoke various local
or systemic mechanisms to help plants sustain their
growth under stress conditions (Yang et al. 2008).
Our results indicated that cyanobacteria when inoc-
ulated in rice caused direct local changes and
enhanced level of phenolic acids, flavonoids and
phytohormones in the root rhizosphere due to
production and release of such metabolites in the
rhizospheric soil. Biologically active substances are
Fig. 6 Peroxidase activity in the leaves of rice plants
inoculated with different cyanobacterial strains 1. Anabaenaoryzae, 2. Anabaena doliolum, 3. Phormidium fragile, 4.
Calothrix geitonos, 5. Hapalosiphon intricatus, 6. Aulosirafertilissima, 7. Tolypothrix tenuis, 8. Oscillatoria acuta, 9.
Plectonema boryanum, 10. Control
Fig. 7 Phenylalanine ammonia lyase (PAL) activity in the
leaves of rice plants inoculated with different cyanobacterial
strains 1. Anabaena oryzae, 2. Anabaena doliolum, 3.
Phormidium fragile, 4. Calothrix geitonos, 5. Hapalosiphonintricatus, 6. Aulosira fertilissima, 7. Tolypothrix tenuis, 8.
Oscillatoria acuta, 9. Plectonema boryanum, 10. Control; One
population t test: t = 22.19803, P = 3.61309E-9, at 0.05
level, the two means are significantly different
Antonie van Leeuwenhoek (2011) 100:557–568 563
123
produced and contained within or confined to the
interior of the cells and are released in the environ-
ment (Sedmak et al. 2009). It is established that
inoculated PGPRs release various kinds of secondary
metabolites as growth promoting substances (Khalid
et al. 2006; Ahmad et al. 2008) and signaling
molecules in the rhizosphere to promote plant growth
(Walker et al. 2003a; Nelson 2004). Phenolics,
especially flavonoids have proven role in plant–
microbe interactions (Peters and Verma 1990) and
enhance root colonization by microbes (Kothandar-
aman et al. 2003), promote allelochemical influence
Table 2 Phenolic acids and flavonoid profile of rhizospheric soil of rice inoculated with different cyanobacterial strains after 15 and
30 days
Treatments Phenolics in rhizospheric soil (lg/g) after 15 days
Phenolic acids Flavonoids
Gallic Gentisic Chlorogenic Ferulic Rutin Quercetin
Anabaena oryzae 60.83 d 0.72 d 6.20 a 0.83 d 0.22 f 0.63 e
Anabaena doliolum 56.23 e 1.75 c 1.09 c-f 1.28 c 0.80 d 1.26 d
Phormidium fragile 108.93 a 2.60 b 0.75 ef 1.30 c 0.57 de 0.53 e
Calothrix geitonos 108.70 a 1.70 c 1.37 cd 0.73 d 0.13 f 0.27 e
Hapalosiphon intricatus 86.63 b 6.67 a 1.60 c 1.13 cd 1.57 c 0.47 e
Aulosira fertilissima 35.17 f 1.77 c 1.07 def 2.51 b 5.93 a 6.77 b
Tolypothrix tenuis 28.10 g 2.40 b 0.67 f 1.03 cd 4.40 b 0.39 e
Oscillatoria acuta 75.60 c 1.70 c 1.22 cde 3.53 a 0.73 d 2.40 c
Plectonema boryanum 108.87 a 1.83 c 2.89 b 0.80 d 0.33 ef 10.30 a
Control 38.63 f 0.97 d 0.97 def 0.24 e 0.20 f 0.70 e
SEM± 1.43 0.176 0.175 0.137 0.108 0.158
CD (P = 0.05) 4.26 0.523 0.52 0.407 0.321 0.469
CV % 3.5 13.8 17.0 17.7 12.6 11.5
Treatments Phenolics in rhizospheric soil (lg/g) after 30 days
Phenolic acids Flavonoids
Gallic Gentisic Chlorogenic Ferulic Rutin Quercetin
Anabaena oryzae 42.40 f 0.50 f 7.26 a 1.62 c 0.04 g 1.17 e
Anabaena doliolum 51.43 e 1.65 e 1.38 d 1.17 d 0.81 d 1.47 d
Phormidium fragile 124.57 c 3.67 c 0.59 f 0.80 e 0.72 de 0.92 f
Calothrix geitonos 138.73 b 1.57 e 1.00 e 0.43 fg 0.04 g 0.74 g
Hapalosiphon intricatus 81.70 d 5.93 b 2.52 c 1.20 d 1.44 c 0.57 h
Aulosira fertilissima 35.73 g 3.17 c 0.87 ef 3.20 b 5.76 a 6.31 b
Tolypothrix tenuis 19.73 h 2.53 d 0.90 ef 1.30 d 4.56 b 0.83 fg
Oscillatoria acuta 82.93 d 2.10 de 1.63 d 4.60 a 0.68 ef 2.57 c
Plectonema boryanum 170.13 a 9.47 a 4.43 b 0.67 ef 0.02 g 9.68 a
Control 51.30 e 0.77 f 0.73 ef 0.33 g 0.58 f 0.73 g
SEM± 1.03 0.172 0.117 0.103 0.041 0.053
CD (P = 0.05) 3.06 0.510 0.348 0.304 0.122 0.1587
CV % 2.2 9.5 9.5 11.6 4.9 3.7
HPLC retention time (Rt) gallic acid- 2.53, gentisic 3.02, chlorogenic 3.56, ferulic 4.32, rutin 2.99 and quercetin 4.98 min
DAI days after inoculation, SEM± standard error of means±, CD critical difference (at significance of 95%), CV coefficient of
variance; Values in the same column followed by a different letter are significantly different (a = 0.05) in Duncan’s multiple range
test
564 Antonie van Leeuwenhoek (2011) 100:557–568
123
Fig. 8 Accumulation of phytohormones in the leaves of rice
plants inoculated with cyanobacterial strains and its correlation
with shoot length. Cyanobacterial strains—1. Anabaenaoryzae, 2. Anabaena doliolum, 3. Phormidium fragile, 4.
Calothrix geitonos, 5. Hapalosiphon intricatus, 6. Aulosirafertilissima, 7. Tolypothrix tenuis, 8. Oscillatoria acuta, 9.
Plectonema boryanum, 10. Control; Two population t- test for
Indole acetic acid (IAA) t = 0.43944, P = 0.66557, at 0.05
level, the two means are not significantly different, Indole
butyric acid: t = -0.66988, P = 0.51312, at 0.05 level, the
two means are not significantly different, shoot length
(30 days): t = 6.30287, P = 6.09732E-6, at 0.05 level, the
two means are significantly different
Fig. 9 Accumulation of phytohormones in the rhizospheric
soil of rice plants inoculated with cyanobacterial strains and its
correlation with root length after 30 days. DAI days after
inoculation, two population t-test: indole acetic acid-
t = 0.335, P = 0.74149, at 0.05 level, the two means are
NOT significantly different; indole butyric acid : t = 0.06934,
P = 0.94578, at 0.05 level, the two means are NOT
significantly different; root length : one population t-test :
t = 20.07489, P = 8.78509E-9, at 0.05 level, the two means
are significantly different. 1. Anabaena oryzae, 2. Anabaenadoliolum, 3. Phormidium fragile, 4. Calothrix geitonos, 5.
Hapalosiphon intricatus, 6. Aulosira fertilissima, 7. Tolypo-thrix tenuis, 8. Oscillatoria acuta, 9. Plectonema boryanum,
10. Control
Antonie van Leeuwenhoek (2011) 100:557–568 565
123
on population of other organisms (Walker et al.
2003a, b) and act as signal molecules (Mandal et al.
2010). In the light of these existing facts, a complex
interactive mechanism due to the presence of metab-
olites may be speculated in the rhizospheric soil of
rice inoculated with the cyanobacterial strains.
PGPRs are also known to create complex interactions
in the rhizosphere (Naher et al. 2009) that favour
chemical diversity, especially of phenolics in the root
exudates (Fletcher and Hedge 1995; Kent and Triplet
2002; Singer et al. 2003; Kothandaraman et al. 2003).
These findings are concurrent with the results on the
enhanced presence of diverse metabolites in the
rhizosphere. Effect of cyanobacterial secondary
metabolites on growth of other algae and higher
plants (Rai et al. 2000) and significant increase in
phenolic level and soil chemical properties following
different doses of inoculation (Inderjit and Keating
1999) is reported and therefore, the cumulative effect
of the metabolites produced and released by the
cyanobacterial inoculants in the rice rhizosphere is
thought to be a major reason responsible for the plant
growth promotion.
Cyanobacterial inoculation also evoked systemic
accumulation of biochemicals (chlorophyll, protein
and total phenol), induced levels of phenylpropanoids
and phytohormones and enhanced enzymatic profile
(peroxidase and PAL) in rice leaves. Many fold
accumulation of phenolics in rice leaves as compared
to control is in concurrence with the earlier reports on
increased level of phenolics in plant tissues following
inoculation with non-pathogenic organisms (Yedidia
et al. 1999). Growth in cyanobacteria-inoculated rice
plants is directly correlated with enhanced systemic
accumulation of metabolites including phytohormones
that are a definite parameter of enhanced growth
(Segura et al. 2009). Also, altered and enhanced status
of peroxidase and PAL in rice leaves following
inoculation with cyanobacteria in comparison to
uninoculated plants may be positively correlated with
the induced systemic tolerance against stress (Lavania
et al. 2006; Yang et al. 2008). A direct correlation
between antioxidant properties and levels of phenolic
acids, flavonoids, PAL and peroxidase enzymes has
been reported earlier (Singh et al. 2009; Gao et al.
2010). Our results indicated that the impact of cyano-
bacterial inoculation on rice plant and rhizosphere soil
under salt stress is similar to the effect of plant growth
promoting rhizobacteria (PGPRs) (Ahmad et al. 2008;
Yang et al. 2008) that are shown to induce systemic
resistance against pathogens due to the induction of
peroxidase (Egley et al. 1983) and PAL enzymes
(Pieterse et al. 1996b), accumulation of phenolics
(Sarma et al. 2002; Singh et al. 2002, 2003; Basha et al.
2006) and plant growth promotion due accumulation of
phytohormones (Khalid et al. 2006; Basha et al. 2006).
Although many attributes of plant growth promoting
traits of cyanobacteria including symbiotic nitrogen
fixation, phosphate solubilization, siderophore pro-
duction and IAA synthesis have been described (Rai
et al. 2000; Fernandez et al. 2000), we conclude that
systemic accumulation of phenylpropanoids in rice
following cyanobacterial inoculation enhanced capa-
bilities of plants for growth and development.
Acknowledgments Authors gratefully acknowledge Indian
Council of Agricultural Research (ICAR), India for financial
support.
References
Ahmad F, Ahmad I, Aqil F, Khan MS, Hayat S (2008)
Diversity and potential of nonsymbiotic diazotrophic
bacteria in promoting plant growth. In: Ahmad I, Pitchel J,
Hayat S (eds) Plant–bacteria interactions: strategies and
techniques to promote plant growth. KGaA, Wiley-VCH,
Verlag Gmbh and Co, Germany, pp 81–109
Barriuso J, Ramos Solano B, Gutierrez Manero FJ (2008)
Protection against pathogen and salt stress by four plant
growth-promoting rhizobcteria isolated from Pinus sp. on
Arabidopsis thaliana. Phytopathology 98:666–672
Basha SA, Sarma BK, Singh DP, Annapurna K, Singh UP
(2006) Differential methods of inoculation of plant
growth-promoting rhizobacteria induce synthesis of phe-
nylalanine-ammonia-lyase and phenolic compounds dif-
ferentially in chickpea. Folia Microbiol 51:463–468
Bloemberg GV, Lugtenberg BJJ (2001) Molecular basis of
plant growth promotion and biocontrol by rhizobacteria.
Curr Opin Plant Biol 4:343–350
Carreno-Lopez R, Campos-Reales N, Elmerich C, Baca BE
(2000) Physiological evidence for differently regulated
tryptophan-dependent pathways for indole-3-acetic acid
synthesis in Azospirillum brasilance. Mol Gen Genet
264:521
Cryl P, Karl G (2008) Secondary metabolites from cyanobac-
teria: complex structures and powerful bioactivities. Curr
Org Chem. 12:326–341
Dixon RA (2001) Natural products and plant disease resis-
tance. Nature 411:843–847
Egley GH, Paul RN, Vaughn KC, Duke SO (1983) Role of
peroxidase in the development of water impermeable
seeds coats in Sida spinosa L. Planta 157:224–232
Fadzilla NM, Finch RP, Burdon RH (1997) Salinity, oxidative
stress and antioxidant responses in shoot cultures of rice.
J Exp Biol 48:325–331
566 Antonie van Leeuwenhoek (2011) 100:557–568
123
Ferjani A, Mustardy L, Sulpice R, Marin K, Suzuki I, Hageman
M, Murata N (2003) Glucosylglycerol, a compatible sol-
ute, sustains cell division under salt stress. Plant Physiol
131:1628–1637
Fernandez VE, Ucha A, Quesada A, Leganes F, Carreres R
(2000) Contribution of N2 fixing cyanobacteria to rice
production: availability of nitrogen from 15N-labelled
cyanobacteria and ammonium sulphate to rice. Plant Soil
221:107–112
Fletcher JS, Hedge RS (1995) Release of phenols by
perennial plant roots and their potential importance
in bioremediation. Environ Toxicol Chem 31:3009–
3016
Gao D, Du L, Yang J, Wu W-M, Hong Liang H (2010) A
critical review of the application of white rot fungus to
environmental pollution control. Crit Rev Biotechnol
30:70–77
Glick B (1995) The enhancement of plant growth by free-
living bacteria. Can J Microbiol 41:109–117
Graham HG (1992) Stabilization of the Prussian blue color in
the determination of polyphenols. J Agri Food Chem
40:801–805
Gutierrez MFJ, Ramos Solano B, Probanja A, Mebouachi J,
Tadeo FR, Talon M (2001) The plant growth-promoting
rhizobcteria Bacillus pumilus and Bacillus licheniformisproduce high amounts of physiologically active gibber-
ellins. Physiol. Plantarum 111:1–7
Inderjit, Keating KI (1999) Allelopathy: principles, procedures,
processes and promises for biological control. In: Sparks
DL (ed) Advances in agronomy. Academic Press, London,
pp 142–207
Karthikeyan N, Prasanna R, Lata N, Kaushik BD (2007)
Evaluating the potential of plant growth promoting cya-
nobacteria as inoculants for wheat. Eur J Soil Biol
43:23–30
Kent AD, Triplet EW (2002) Microbial communities and their
interactions in soil and rhizosphere ecosystems. Annu Rev
Microbiol 56:211–236
Khalid A, Arshad M, Zahir A (2006) Phytohormones: micro-
bial production and applications. In: Uphoff N (ed) Bio-
logical approaches to sustainable soil systems. CRC Press,
London, pp 207–220
Khan ZUM, Tahmida Begum ZN, Mandal R, Hossain MZ
(1994) Cyanobacteria in rice soils. World J Microbiol
Biotechnol 10:296–298
Kloepper JW, Scrhoth MN, Miller TD (1980) Effects of rhi-
zosphere colonization by plant growth-promoting rhizo-
bacteria on potato plant development and yield.
Phytopathology 70:1078–1082
Kothandaraman N, Chanbasha B, Vladimir BB, Swarup S
(2003) Enhancement of plant-microbe interactions using a
rhizosphere metabolomics-driven approach and its appli-
cation in the removal of polychlorinated biphenyls. Plant
Physiol 132:146–153
Kumar A, Singh DP, Tyagi MB, Kumar A, Prasuna EG,
Thakur JK (2000) Production of hepatotoxin by the cya-
nobacterium Scytonema sp. Strain BT 23. J Microbiol
Biotechnol 10:375–380
Lavania M, Chauhan PS, Chauhan SVS, Singh HB, Nautiyal
CS (2006) Induction of plant defense enzymes and
phenolics by treatment with plant growth—promoting
rhizobacteria Serratia marcescens NBRI1213. Curr
Microbiol 52:363–368
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Pro-
tein measurement with folin phenol reagent. J Biol Chem
193:265–275
Lugtenberg B, Kamilova F (2009) Plant-growth promoting
rhizobacteria. Annu Rev Microbiol 63:541–556
Mandal SM, Chakraborty D, Dey S (2010) Phenolic acids act
as signalling molecules in plant-microbe symbioses. Plant
Signal Behav 5:359–368
Moon CD, Giddens SR, Zhang X-X, Jackson RW (2008)
Molecular mechanisms underpinning plant colonization by
a plant growth promoting rhizobacterium. In: Ahmad I,
Pitchel J, Hayat S (eds) Plant–bacteria interactions: strat-
egies and techniques to promote plant growth. KGaA,
Wiley-VCH, Verlag Gmbh and Co., Germany, pp 111–128
M’Piga P, Belanger RR, Paulitz TC, Benhamou N (1997)
Increased resistance to Fusarium oxysporum f. sp. radicis-
lycopersici in tomato plants treated with endophytic bac-
terium Pseudomonas fluorescens strain 63–28. Physiol
Mol Plant Pathol 50:301–320
Naher UA, Othman R, Shamsuddin ZHJ, Saud HM, Ismail MR
(2009) Growth enhancement and root colonization of rice
seedlings by Rhizobium and Corynebacterium spp. Int J
Agric Biol 11:586–590
Nelson LM (2004) Plant growth promoting rhizobacteria
(PGPR): prospects for new inoculants. Crop Management
doi: 10.1094/CM-2004-0301-05-RV
Niranjan A, Barthwal J, Lehri A, Singh DP, Govindrajan R,
Rawat AKS, Amla DV (2009) Development and valida-
tion of an HPLC-UV-MS–MS method for identification
and quantification of polyphenols in Artemisia pallens L.
Acta Chromatogr 21:105–116
Peters NK, Verma DPS (1990) Phenolic compounds as regu-
lators of gene expression in plant-microbe interactions.
Mol Plant Microbe Interact 3:4–8
Pieterse CMJ, van Wees SCM, van Pelt JA, Trijssenaar A,
Van’t Westende YAM, Bolink EM, van Loon LC (1996a)
Systemic resistance in Arabidopsis thaliana induced by
biocontrol bacteria. Meded Fac Land bouwkd Toegep
Biol Wet Univ Gent 61:209–220
Pieterse CMJ, van Wees SCM, Hoffland E, van Pelt JA, van
Loon LC (1996b) Systemic resistance in Arabidopsisinduced by biocontrol bacteria is independent of salicylic
acid accumulation and pathogenesis-related gene expres-
sion. Plant Cell 8:1225–1237
Rai AN, Soderback E, Bergman B (2000) Cyanobacterial-plant
symbioses: a review. New Phytol 147:449–481
Ramos Solano B, Barriuso Maicas J, Pereyra de la Iglesia MT,
Domenech J, Gutierrez Manero FJ (2008) Systemic dis-
ease protection elicited by plant growth promoting rhi-
zobacteria strains: relationship between metabolic
responses, systemic disease protection, and biotic elici-
tors. Biol Control 98:451–457
Rastogi RP, Sinha RP (2009) Biotechnological and industrial
significance of cyanobacterial secondary metabolites.
Biotechnol Adv 27:521–539
Rodriguez-Diaz M, Rodelas-Gonzales B, Pozo-Clemente C,
Martinez-Toledo MC, Gonzalez-Lopez J (2008) A review
of the taxonomy and possible screening traits of plant
growth promoting rhizobacteria. In: Ahmad I, Pitchel J,
Antonie van Leeuwenhoek (2011) 100:557–568 567
123
Hayat S (eds) Plant–bacteria interactions: strategies and
techniques to promote plant growth. KGaA, Germany-
Wiley-VCH, Verlag Gmbh and Co, Germany, pp 55–80
Sarma BK, Singh DP, Mehta S, Singh HB, Singh UP (2002)
Plant growth-promoting rhizobacteria-elicited alterations
in phenolic profile of chickpea (Cicer arietinum) infected
by Sclerotium rolfsii. J Phytopathol 150:277–282
Sedmak B, Carmeli S, Pompe-Novak M, Tusek-Znidaric M,
Grach-Pogrebinski O, Elersek T, Zuzek MC, Bubik A,
Frangez R (2009) Cyanobacterial cytoskeleton immuno-
staining: the detection of cyanobacterial cell lysis induced
by planktopeptin BL1125. J Plankton Res 31:1321–1330
Segura A, Rodriguez-Conde S, Ramos C, Ramos JL (2009)
Bacterial responses and interactions with plants during
rhizoremediation. Microbial Biotechnol 2:452–464
Senaratna T, McKersie BD, Borochov A (1987) Desiccation
and free radical mediated changes in plant membranes.
J Exp Bot 38:2005–2014
Senaratna T, Touchell D, Bunn E, Dixon K (2000) Acetyl
salicylic acid (aspirin) and salicylic acid induced multiple
stress tolerance in bean and tomato plants. Plant Growth
Regul 30:157–161
Singer AC, Crowley DE, Thompson IP (2003) Secondary plant
metabolites in phytoremediation and biotransformation.
Trends Biotechnol 21:123–130
Singh UP, Sarma BK, Singh DP, Bahadur A (2002) Plant
growth-promoting rhizobacteria-mediated induction of
phenolics in pea (Pisum sativum) after infection with
Erysiphe pisi. Curr Microbiol 44:396–400
Singh UP, Sarma BK, Singh DP (2003) Effect of plant growth-
promoting rhizobacteria and culture filtrate of Sclerotiumrolfsii on phenolic and salicylic acid contents in chickpea
(Cicer arietinum). Curr Microbiol 46:131–140
Singh BN, Singh BR, Singh RL, Prakash D, Singh DP, Sarma
BK, Upadhyay G, Singh HB (2009) Polyphenolics from
various extracts/fractions of red onion (Allium cepa) peel
with potential antioxidants and antimutagenic activities.
Food Chem Toxicol 47:1161–1167
Stanier RY, Kunisawa R, Mandel M, Cohen-Bazire G (1971)
Purification and properties of unicellular blue-green algae
(order Chroococcales). Bacteriol Rev 35:171–205
Vaishampayan A, Sinha RP, Haider D-P, Dey T, Gupta AK,
Bhan U, Rao AL (2001) Cyanobacterial biofertilizers in
rice agriculture. Bot Rev 67:453–516
Walker TS, Bais HP, Grotewold E, Vivanco JM (2003a) Root
exudation and rhizosphere biology. Plant Physiol 132:
44–51
Walker TS, Bais HP, Halligan KM, Stermitz FR, Vivanco JM
(2003b) Metabolic profiling of root exudates of Arabid-opsis thaliana. J Agri Food Chem 41:2548–2554
Wink M, Schimmer O (1999) Modes of action of defence
secondary metabolites. In: Wink M (ed) Functions of plant
secondary metabolites and their exploitation in biotech-
nology. CRC Press, Boca Raton, Florida, pp 17–112
Yandigeri MS, Yadav AK, Meena KK, Pabbi S (2010) Effect of
mineral phosphates on growth and nitrogen fixation of dia-
zotrophic cyanobacteria Anabaena variabilis and Westiell-opsis prolifica. Antonie van Leeuwenhoek. 97:297–306
Yang J, Kloepper JW, Ryu C-M (2008) Rhizosphere bacteria
help plants tolerate abiotic stress. Cell Press. doi:10.1016/
j.tplants.2008.10.0
Yedidia I, Benhamou N, Chet I (1999) Induction of defense
responses in cucumber plants (Cucumis sativus L.) by the
biocontrol agent Trichoderma harzianum. Appl Environ
Microbiol 65:1061–1070
Yedidia I, Shoresh M, Kerem Z, Benhamou N, Kapulnik Y,
Chet I (2003) Concomitant induction of systemic resis-
tance to Pseudomonas syringae pv. lachrymans in
cucumber by Trichoderma asperellum (T-203) and accu-
mulation of phytoalexins. Appl Environ Microbiol
69:7343–7353
568 Antonie van Leeuwenhoek (2011) 100:557–568
123