art%3A10.1007%2Fs10482-011-9611-0

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: [email protected]

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


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.


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


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


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.


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


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


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


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


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


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,


123

http://dx.doi.org/10.1094/CM-2004-0301-05-RV

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


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


123

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