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Antonie van Leeuwenhoek (2011) 100:557–568

DOI 10.1007/s10482-011-9611-0

ORIGINAL PAPER

Cyanobacteria-mediated phenylpropanoids
and phytohormones in rice (Oryza sativa) enhance plant
growth 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
Ó 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, Phormidium 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 compounds 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 phenylpropanoids in plant leaves led us to conclude that
cyanobacterial inoculation correlates positively with

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

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 potentially 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 (Ferna´ndez 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 rhizobacteria (PGPRs) (Kloepper et al. 1980) play critical
role in plant health and nutrition (Ahmad et al.
2008). They can benefit plant growth by improving

123

India. doliolum. 1971) in 100 ml flasks. Cryl and Karl 2008. Moon et al. 2000).0 ml cyanobacterial cell suspension (OD663nm * 0. Plant growth assessment Ten rice plants per treatment and control were randomly harvested from the pots after 15 and 30 days of inoculation. It is widely realized that in plants. EC 5. Rice seeds (variety UPR 1823) were obtained from the Directorate of Seed Research. 123 Antonie van Leeuwenhoek (2011) 100:557–568 Materials and methods The microorganisms. Rhizobacteria-elicited ‘‘induced systemic tolerance’’ (IST) that can enhance tolerance in plants to abiotic stress due to physical and chemical changes (Yang et al. 1987) and root colonization with PGPRs facilitate plants to fight against pathogen or abiotic stress mediated losses (Barriuso et al. 24 plants per treatment) and harvested at 15 and 30 days. 2008) is a relatively recent approach that considerably overlaps with the mechanisms of systemic induced resistance in plants (Ramos Solano et al. Bloemberg and Lugtenberg 2001). w/w) and pots were transferred to the culture room maintained with fluorescent light at 25 ± 2°C. growth conditions and seed treatment Cyanobacterial strains namely Anabaena oryzae. producing plant growth regulators (Gutierrez et al. Sarma et al. Maunath Bhanjan. Plants were allowed to grow in four replications (6 plants per pot. Release of a diverse array of biologically active metabolites by the growing cyanobacterial cells (Kumar et al. 2001). In present communication. Surface sterilized seeds were pre-soaked in DDW and kept on sterilized moist Whatman filter paper one day before the inoculation was made. All the strains were transferred from their respective slants in 40 ml (93) of BG11 medium (Stanier et al. Barriuso et al. 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 1.67) and left for 4 h. 2008. 2008).) filled with sterile soil (pH 8. Thirty seeds were transferred to glass tubes containing 5. PAL) status of rice in planta under stressed soil and in the root rhizosphere.b. 2008). Aulosira fertilissima. inducing systemic resistance against pathogenic attack (Ahmad et al. Dixon 2001. Yedidia et al. evoking changes in metabolic status of plants (Pieterse et al. Since cyanobacteria naturally colonize rice roots in salt affected soils.0% carboxymethylcellulose (CMC) as binder. Singh et al. rhizoremediation and plant stress control (Lugtenberg and Kamilova 2009). 2008. 1997. Rastogi and Sinha 2009) in the rhizosphere soil may also assist in enhancing plant growth in salt stressed soils. 2008. Inoculated seeds were sown in sterilized plastic pots (6 seeds per pot of 15 cm dia. 2003). Calothrix geitonos.2 dS/ m) containing sand (3:1. Plants maintained in similar growth conditions but remained uninoculated served as control. Wink and Schimmer 1999.558 plant nutrition and soil fertility (Glick 1995. Root and shoot length and fresh weight of each and every plant were measured immediately after harvesting. 2002. . it is imperative to suggest their role as PGPR and therefore. 2003. India.8. M’Piga et al. Cultures were bubbled with air containing 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. the physiological disorders due to abiotic stresses or pathological disorders caused by microbial agents that promote the development of hypersensitive reactions. usually involve destructive free radical-mediated oxidative degradation of biomolecules (Senaratna et al. Oscillatoria acuta and Plectonema boryanum were obtained from the NAIMCC culture collection. we report impact of cyanobacterial colonization on the physical growth. 2002. A. 2000. 2008. 1996a. Maunath Bhanjan. Rodriguez-Diaz et al. 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. Hapalosiphon intricatus. Tolypothrix tenuis. Phormidium fragile. metabolic (phenylpropanoids and phytohormones) and enzymatic (peroxidase and phenylalanine ammonia lyase. Studies suggest that plants defend themselves from such oxidative damages by the changes in their physiological and biochemical status (Senaratna et al. Lugtenberg and Kamilova 2009).

cooled and centrifuged at 150009g for 5 min.8) containing 1 mM EDTA and 2% (w/v) polyvinylpyrrolidone. 100 mg/l) and the homogenates were kept overnight at 4°C in dark. The increase in the absorbance due to oxidation of guaiacol was measured at 470 nm (E = 26. Reverse phase liquid chromatographic analysis of the samples (injection volume 10 ll) 123 . Phenolics were extracted from the freshly harvested rice leaves as described earlier (Singh et al. (2003). (2003). The supernatants were pooled together and solvent was evaporated to dryness. v/v. 0. The resultant was redissolved in HPLC grade methanol for the estimation of phytohormones. the resulting supernatant was frozen at -20°C. The supernatant was quantified for total content of protein as per the method of Lowry et al. Metabolic profiling of rhizospheric soil containing root exudates (Walker et al. 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. 2003). 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. India) as standard.1 mM EDTA. thawed and centrifuged at 90009g for 30 min at 4°C to remove impurities. For the extraction of phytohormones.6 mM-1cm-1). Total soluble phenol (TSP) was estimated spectrophotometrically using the Prussian blue method as described by Graham (1992) and expressed in terms of gallic acid equivalents by using gallic acid (HiMedia. The reaction mixture (total volume 2 ml) contained 25 mM sodium phosphate buffer (pH 7. After re-extraction (93) with 80% methanol. PAL activity was estimated from the same enzyme extract as per the method described by Singh et al. The pellet was resuspended in 1 N NaOH (5 ml). Briefly. USA) equipped with binary Waters 515 reciprocating pumps. boiled for 30 min. chlorophyll in the supernatant was quantified in terms of A665nm (Ferjani et al. one g fresh leaves were homogenized with 80% methanol containing butylated hydroxytoulene (BHT. 2003). v/v). The dried extract was redissolved in methanol (HPLC grade) and subjected to filtration prior to analysis. After removal of the precipitate through centrifugation at 150009g for 5 min. a variable photodiode array (PDA) detector (Waters 2996) and system controller equipped with TM WatersÒEmpower software for data integration and analysis. Total protein content (TPC) was extracted as per the method described by Ferjani et al. The clear supernatant thus obtained was filtered and the solvent was evaporated under vacuum. The clear greenish supernatant was collected and the cell debris was again suspended in extraction solvent and kept for 4 h. (1951) with bovine serum albumin (BSA) as standard.5 g freshly harvested leaf tissues using methanol: water (9:1. (1983).05% guaiacol (2-ethoxyphenol). 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. 1. 0. Peroxidase activity was measured using a modified procedure of Egley et al.Antonie van Leeuwenhoek (2011) 100:557–568 559 Biochemical tests Extraction of phenolics and phytohormones Chlorophyll content in rice leaves was quantified by extracting the pigment from 0. Finally the supernatants were pooled and thoroughly mixed with a pinch of charcoal to remove the pigments. Dried samples were redissolved in HPLC grade methanol by vortexing and stored at 4°C for further analysis. 2003b) from each treatment was carried out by drying one g soil under vacuuo and suspending the same in methanol : water (1:1. HPLC analysis High performance liquid chromatography (HPLC) of rice leaves and rhizospheric soil extracts was performed using HPLC system (Waters. 5 ml) thrice.0 mM H2O2 and 100 ll enzyme extract.0). Analytical grade reagents were used throughout the experiments.05 M sodium phosphate buffer (pH 7. Absorbance was recorded at 700 nm using UV–VIS spectrophotometer (Shimadzu Corporation. 1 g leaf tissues was macerated in a pestlemortar and then mixed with 5 ml of extraction solvent methanol: water (1:1. The supernatant used as enzyme extract was obtained from the homogenate by centrifugation at 130009g for 15 min at 4°C. Japan).

3. Growth promotion effect as well as the colonization of soil and plant root by a potential cyanobacterial strain is also shown in Fig. Applied Biosystems. Maximum shoot and root length and plant weight (wt) (17. India. Oscillatoria acuta. 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. Tolypothrix tenuis. Consistent systemic accumulation of phenolic acids (gallic.3 cm and 2.45 lm membrane filter prior to injection in the sample loop. 9. 4. Results were reported as mean (±) standard deviation (SD) of four replicates from pot experiments and three from sample analysis using HPLC. 2. 4a and b. The Fig. In comparison to the uninoculated (control) plants. 7. tenuis after 30 days of inoculation (Fig. doliolum. 1. strains—1.0183. 8.34 mg/g in P. caffeic acid (179/135). the two means are significantly different 123 Antonie van Leeuwenhoek (2011) 100:557–568 compounds were detected according to their respective m/z values of their parent and product ions.d. Differences were considered to be significant at the 95% confidence level. boryanum and total protein 9. 7. intricatus inoculated plant leaves could not be traced after . 2. boryanum. 2). (2000) at a flow rate of 1 ml/min of methanol: 1% aqueous acetic acid (24:76. ferulic. caffeic. two population t-test: shoot length t = 5.43 mg/g in A.2 mg/g fresh wt in P. v/v) and detection at 254 and 280 nm for phenolic acids gallic. Calothrix geitonos. Canada) as per the methods described earlier (Singh et al. Flavonoids (rutin and quercetin) and phytohormones (indole acetic and indole butyric acid) were analyzed as per the method of Carreno-Lopez et al.0. 2009. India) were used throughout the analysis. A. Plectonema boryanum.05 level. chlorophyll and total protein in rice leaves varied significantly within the plants inoculated with different cyanobacterial strains (Fig. chlorogenic acid (353/191)..6 cm. gallic acid (169/125). ferulic acid (193/134) and quercetin (301/151). oryzae to 143. v/v) as mobile phase. chlorogenic. 5. Hapalosiphon intricatus. 5 lm particle size) at 25 ± 1°C at a flow rate of 1 ml/min of the mobile phase methanol: 0. P = 0. all the treatments performed significantly well in terms of the physical and biochemical growth indicators (Figs. P = 1.77. chlorogenic and ferulic acids) was observed in the leaves of inoculated plants after 15 and 30 days of growth (Table 1). Control. Chlorophyll content in plant leaves ranged from 57. Quantitative profile of different biochemicals viz. Anabaena oryzae. 3).560 was carried out in isocratic mode on a C-18 column (250 9 4.79978E-5. 1 Effect of cyanobacterial inoculation on shoot and root length of rice. Samples were subjected to membrane filtration through 0.18 g after 15 days and 24. gentisic and cinnamic acids.86 mg/g fresh wt in T. API 2000.1 g after 30 days. Niranjan et al. root length t = 2. 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. 6.59.6 cm and 3. 3). at 0. fragile to 17. fertilissima inoculated plant leaves and ferulic acid in H. In certain treatments the presence of chlorogenic acid in A.4% acetic acid in water (60:40. Results Rice plants inoculated with different strains of cyanobacteria showed differential responses in terms of shoot and root length and plant weight (Figs. 2009). HPLC grade solvents and chemicals (E Merck and Hi Media.6 mm i. 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. Phormidium fragile. respectively) was observed in the plants inoculated with P. Ontario. Aulosira fertilissima. 10.. 8. 3). 1.1 cm.

0 Plant fresh wt (g) 2. the two means are significantly different and ferulic acids (111. 6. doliolum. P = 0. 9. Phormidium fragile. 3 Quantitative profile of different biochemicals (total protein and chlorophyll) in leaves of rice plants inoculated with different cyanobacterial strains— 1. acuta 123 . Plectonema boryanum. A. 10. two population t-test for total protein t = 6. 7. Total protein in terms of bovine serum albumin.4. Aulosira fertilissima. 6. 3.50 and 3. However. A. Calothrix geitonos. 4.0 1. Oscillatoria acuta. Calothrix geitonos. 200 total protein after 15 days total protein after 30 days Chl content after 15 days Chl content after 30 days 180 Quantity (mg/g fresh wt) 160 140 120 100 80 60 40 20 0 0 1 2 3 4 5 6 7 8 9 10 cyanobacterial strains Fig. 2 Effect of cyanobacteria inoculation on fresh weight of rice plants. and b root colonization with O. Results on peroxidase (Fig. Tolypothrix tenuis. doliolum.97 lg/g fresh wt) was recorded in rice leaves inoculated with H. 4 a Colonization of inoculated cyanobacterium Oscillatoria acuta on the soil surface. A. strains—1. chlorogenic Fig.00558. 8. Tolypothrix tenuis. Anabaena oryzae. acuta and P.05 level. acuta respectively. 8.27.05 level. doliolum inoculated plant leaves. Anabaena oryzae. 3.62. 10.00117.14677.99046E-6. oryzae and O. 5). Control. doliolum. P. 4. 5. 6. the two means are significantly different 15 days of inoculation. after 30 days of inoculation.5 1. acuta. Plants inoculated with O. the in planta accumulation was 144. 5.60 and 9. Aulosira fertilissima. Almost similar trend was observed with both the enzymes. 9. Phormidium fragile. 8. Chlorophyll content: t = 3. maximum accumulation of gallic.63 lg/g fresh wt respectively in O. at 0. boryanum. A. caffeic. at 0. It is evident that 30 days after inoculation favored total phenol content in rice leaves. Plectonema boryanum. P= 1. two population t-test: Plant wt t = 3. although the compounds appeared in the leaves after 30 days.5 2. Oscillatoria acuta.87111. 5. P = 0. 6) and PAL activity (Fig. Hapalosiphon intricatus. After 15 days.85. 2. 7) in the leaves of the plants inoculated with cyanobacterial strains showed enhanced enzyme activity after 15 and 30 days. Rice leaves showed maximum accumulation of total phenol following inoculation with A.Antonie van Leeuwenhoek (2011) 100:557–568 561 After 15 days of inoculation After 30 days of inoculation 3.7. intricatus. Control.0 0 1 2 3 4 5 6 7 8 9 10 Cyanobacterial strains Fig. A. oryzae (Fig. Hapalosiphon intricatus. boryanum showed high peroxidase and PAL activity while all other treatments induced enzyme activity as compared to control. 3. oryzae and A. 7. 2.

2 12.60 a 2.23 c 3.6 8.32 min DAI days after inoculation.70 b Calothrix geitonos 61.4 a 1. Calothrix geitonos.63 j 1.98 e 2.50 b 1.56.32 3. 3.70 b 1. 8.27 a 5.05 level.97 a 144. CV coefficient of variance.17 d 1. P.4 12.298 0.73 d 0. SEM± standard error of means±.53 b 1.73 d 0.84 g 0.50 ef 2. caffeic 3.70 d 3.63 f 5. the two means are significantly different Phenolic acids (gallic.67 c Anabaena doliolum 34.83 d Aulosira fertilissima 75.27 e 1.63 e 0. One population t-test for total phenol : t = 2.42 1. 9. 5.63 a Phormidium fragile 28.93 fg 0. Tolypothrix tenuis. boryanum inoculation showed maximum gallic acid content (170.50 a 1.30 d 3.16 b 2.44946. Anabaena oryzae.63 b 52.62 de 1.54 de 0.8 HPLC retention time (Rt) gallic acid 2.80 d SEM± CD (P = 0. P = 0.50 c 84.50 c 2.83 c 3.100 0.07 e 6.10 c 1.30 de 3.63 b 9. at 0.22.40 d 4.13 lg/g) after 30 DAI in the rhizosphere soil followed by gentisic acid (9.00 c 1.70 a 3.17 h 4.551 0.37 de 2. maximum content of chlorogenic acid (7.57 b 2.00 g 2.60 b 1.16 e 2.62 a 1.60 cd 2. Plectonema boryanum.184 0.93 0.37 e 1.00 f 0. gentisic. Oscillatoria acuta.6 28.5 1.40 ef 67.13 de 2.47 lg/ g).57 e 1.26 lg/g) was found in rhizosphere soil of 123 .13 d Oscillatoria acuta 90.03 i 2. 10. 6.872 2.82 e 0.57 bc 0.53. Values in the same column followed by a different letter are significantly different (a = 0.7 9.549 0.80 de 109. Control.80 a Plectonema boryanum 87.27 h 1.02477.00 fg 135.43 f 0.60 b Tolypothrix tenuis 56.431 CV % 3. 7.40 b 1.05) 1. Similarly. Phormidium fragile.37 efg 2. ferulic 4. 2.578 0.43 b 4. 5 Total phenol content in terms of gallic acid equivalents in the leaves of rice plants inoculated with different cyanobacterial strains—1. Aulosira fertilissima.23 b 8.195 0.05) in Duncan’s multiple range test Fig.37 cd 129.63 c 3.562 Antonie van Leeuwenhoek (2011) 100:557–568 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 75.23 0.67 c Control 20.90 de 45.76 f 6. CD critical difference (at significance of 95%). chlorogenic 3.70 g 88.8 12.80 f 0.83 d Hapalosiphon intricatus 111. 4.47 e 1.17 g 2.50 g 22. Anabaena doliolum.21 efg 1.59 0. 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).185 0. Hapalosiphon intricatus.145 0.

flavonoids and phytohormones in the root rhizosphere due to production and release of such metabolites in the rhizospheric soil. Overall. fertilissima and quercetin (9.68 lg/g) in P. 9. 9). Biologically active substances are 123 . 10. Aulosira fertilissima. and protein content) of rice grown under stress soil (pH 8.Antonie van Leeuwenhoek (2011) 100:557–568 Fig. chlorophyll. Aulosira fertilissima. 1997). Phormidium fragile. 3. 4. 9) inoculated with different cyanobacterial strains. However. its presence was directly correlated with the root or shoot length. 7 Phenylalanine ammonia lyase (PAL) activity in the leaves of rice plants inoculated with different cyanobacterial strains 1. 3. Anabaena doliolum. 6. 6. 8) and the rhizospheric soil (Fig. boryanum showed high content of phytohormones that was reflected in terms of root and shoot length. Control Fig. the level of phytohormones in the rhizospheric soil was also fairly high and corresponded with root length (Fig.2 dS/m). the two means are significantly different A. Hapalosiphon intricatus. Control. The level of phytohormones (IAA and IBA) was determined in rice leaves (Fig.19803. Anabaena doliolum. 1). inoculation of rice plants with P. EC 5. 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. increased levels of phenolics. 8. plant height showed increasing trend except in certain treatments (Fig. 4. 2008). 7. 9. Plectonema boryanum. 10. One population t test: t = 22. Although the presence of IAA was more prevalent quantitatively than the IBA. Oscillatoria acuta. 2. at 0. Reduced growth and development of rice plants due to salt stress in terms of damaged biochemical and physiological mechanisms is documented (Fadzilla et al.61309E-9. Inoculation of cyanobacterial strains favored enhanced content of compounds in comparison to control. 5. Tolypothrix tenuis. Phormidium fragile. 5. Simultaneously. Oscillatoria acuta. enhanced content of total phenol.76 lg/g) in A. Anabaena oryzae. Interestingly. Calothrix geitonos. acuta. The level of phytohormones in plant leaves was very high as compared to control and so was the plant height.05 level. Hapalosiphon intricatus. 8.60 lg/g) in O. Calothrix geitonos. Results indicated systemic accumulation of phenylpropanoid metabolites. shoot length. 7.8. Anabaena oryzae. Tolypothrix tenuis. flavonoids and phytohormones in the root rhizosphere were also observed. 6 Peroxidase activity in the leaves of rice plants inoculated with different cyanobacterial strains 1. biomass accumulation. P = 3. Corresponding to the high levels of IAA and IBA in leaves. 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. Our results indicated that cyanobacteria when inoculated in rice caused direct local changes and enhanced level of phenolic acids. peroxidase and PAL enzyme and induced accumulation of phytohormones in plant leaves. Plectonema boryanum. ferulic acid (4. Qualitative and quantitative analysis of the 563 compounds was performed by reverse-phase HPLC and further validated by the LC-MS/MS analysis. oryzae. 2. boryanum inoculated plants (Table 2). rutin (5.

5 13.13 f 0.30 e 0. chlorogenic 3.63 b 6. It is established that inoculated PGPRs release various kinds of secondary metabolites as growth promoting substances (Khalid et al.103 0.17 e Anabaena doliolum 51.5 Treatments Phenolics in rhizospheric soil (lg/g) after 30 days Phenolic acids Flavonoids Gallic Gentisic Chlorogenic Ferulic Rutin Quercetin Anabaena oryzae 42.348 0.57 c 3.83 c 2.73 d 2.67 a 1.57 c 0.122 0. 2008) and signaling 123 molecules in the rhizosphere to promote plant growth (Walker et al.00 e 2.5 9.60 c 1.17 f 28.72 de 0. promote allelochemical influence .05) 4.43 b 0.17 d 0.73 h 2.23 e 1.52 c 0.73 b 81.7 HPLC retention time (Rt) gallic acid.40 b 6.77 c 2.27 e Hapalosiphon intricatus 86.99 and quercetin 4.68 ef 2.07 def 0.304 0.40 c Plectonema boryanum 108.52 0.83 d 0.041 0.89 b 0.158 CD (P = 0.58 f 0.9 3.73 g 3. CD critical difference (at significance of 95%).321 0.53.175 0.68 a Control 51.2. rutin 2.43 e 1.72 d 6.053 CD (P = 0.73 ef 0.26 d Phormidium fragile 108. Phenolics.63 d 4.05) in Duncan’s multiple range test produced and contained within or confined to the interior of the cells and are released in the environment (Sedmak et al.47 a 4.80 d 1.57 de 0.50 f 7.93 a 4.62 c 0.57 e 5.13 a 9. Nelson 2004).83 d 0.63 f 0.03 cd 5.117 0.76 a 6.60 b 0.40 f 0. gentisic 3. 2009).47 e Aulosira fertilissima Tolypothrix tenuis 35.04 g 1.53 a 0.47 d Phormidium fragile 124.40 b 1.31 b Tolypothrix tenuis 19.564 Antonie van Leeuwenhoek (2011) 100:557–568 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 Gallic Flavonoids Gentisic Chlorogenic Ferulic Rutin Quercetin Anabaena oryzae 60.108 0.44 c 0.93 b 1.80 d 0.1587 CV % 2.30 d 4.20 b 5.70 a 1.70 c 1.30 c 0.04 g 1.02 g 9.60 a 0.81 d 1.93 a 2.02.06 0. especially flavonoids have proven role in plant– microbe interactions (Peters and Verma 1990) and enhance root colonization by microbes (Kothandaraman et al.97 def 0.5 11.33 ef 10.0 17.70 d 1.75 c 1.37 cd 0.70 c 1. Ahmad et al.53 d 0.39 e Oscillatoria acuta 75.43 0.8 17.22 f 0.77 b 0.73 d 0.57 c Plectonema boryanum 170.24 e 0.56.59 f 0.10 de 1.7 12.09 c-f 1.77 f 0.17 c 0.22 cde 3.75 ef 1.26 a 1.92 f Calothrix geitonos Hapalosiphon intricatus 138.67 ef 0.38 d 1. SEM± standard error of means±.20 d 0.74 g 0.176 0.98 min DAI days after inoculation.56 b 0.87 a 1. 2003).43 fg 1.20 a 0.469 CV % 3.13 cd 1.87 ef 3.63 e Anabaena doliolum 56. Values in the same column followed by a different letter are significantly different (a = 0.407 0.57 h Aulosira fertilissima 35.137 0. CV coefficient of variance.73 g SEM± 1.172 0.51 b 1.60 c 1.33 g 0.53 e Calothrix geitonos 108.97 d 0. 2006. ferulic 4.90 ef 1.26 0.10 g 1.523 0.67 f 2.510 0.05) 3.32. 2003a.30 a Control 38.80 e 0.67 c 0.65 e 1.20 f 0.6 4.2 9.93 d 2.83 fg Oscillatoria acuta 82.6 11.03 0.28 c 0.70 e SEM± 1.

Calothrix geitonos. Anabaena doliolum. at 0.66988. 6. the two means are significantly different. Tolypothrix tenuis. Oscillatoria acuta. 8 Accumulation of phytohormones in the leaves of rice plants inoculated with cyanobacterial strains and its correlation with shoot length. at 0. 9. Plectonema boryanum.05 level. Plectonema boryanum.09732E-6. 4. 5.06934. Control. 9 Accumulation of phytohormones in the rhizospheric soil of rice plants inoculated with cyanobacterial strains and its correlation with root length after 30 days.05 level. P = 0. at 0. the two means are not significantly different. Anabaena oryzae.74149. Control 123 .05 level. root length : one population t-test : t = 20. P = 0.test for Indole acetic acid (IAA) t = 0.78509E-9. at 0.05 level. Anabaena doliolum. Phormidium fragile. 8. 1. shoot length (30 days): t = 6.07489. Aulosira fertilissima. 10.05 level.05 level. Calothrix geitonos. P = 6.43944. Phormidium fragile. P = 8. the two means are significantly different Fig. 3.94578. 5.Antonie van Leeuwenhoek (2011) 100:557–568 565 Fig. P = 0. Oscillatoria acuta.335. Hapalosiphon intricatus. 8.51312. 7. Cyanobacterial strains—1. Anabaena oryzae. 2. 7. Aulosira fertilissima. Two population t. indole butyric acid : t = 0. the two means are not significantly different.66557.30287. 10. Tolypothrix tenuis. DAI days after inoculation. P = 0. Hapalosiphon intricatus. 9. Indole butyric acid: t = -0. the two means are NOT significantly different. 6. the two means are NOT significantly different. 3. at 0. two population t-test: indole acetic acidt = 0. at 0. 4. 2.

we conclude that systemic accumulation of phenylpropanoids in rice following cyanobacterial inoculation enhanced capabilities of plants for growth and development. oxidative stress and antioxidant responses in shoot cultures of rice. flavonoids. 2008). Verlag Gmbh and Co. 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. 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. Kent and Triplet 2002. 2006). Yang et al. Curr Opin Plant Biol 4:343–350 Carreno-Lopez R. Curr Org Chem. Effect of cyanobacterial secondary metabolites on growth of other algae and higher plants (Rai et al. 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