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Journal of soil science and plant nutrition

versión On-line ISSN 0718-9516

J. Soil Sci. Plant Nutr. vol.11 no.3 Temuco  2011 

Journal of Soil Science and Plant Nutrition, 2011, 11 (3), 1-12


Indole acetic acid and phytase activity produced by rhizosphere bacilli as affected by pH and metals


J. J. Acuña2, M.A. Jorquera1*, O. A. Martínez2, D. Menezes-Blackburn2, M. T. Fernández3, P. Marschner4, R. Greiner5 and M. L. Mora1

1Center of Plant, Soil Interaction and Natural Resources Biotechnology, Scientific and Biotechnological Biore-source Nucleus, Av. Francisco Salazar 01145, Universidad de La Frontera, Temuco, Chile, E-mail:;
2Programa de Doc-torado en Ciencias de Recursos Naturales, Universidad de La Frontera, Temuco, Chile;
3Instituto Cubano de Investigacion de los Derivados de la Caha de Azucar, Ciudad de La Habana, Cuba.
4School of Agriculture, Food and Wine, The University of Adelaide, Australia.
5Department of Food Technology and Bioprocess Engineering, Max Rubner-Institut, Federal Research Institute of Nutrition and Food, Karlsruhe, Germany. Corresponding author: Tel.: +56 (45) 325 467; fax: +56 (45) 325 053;


The abilities to produce indole acetic acid (IAA) and mineralize organic phosphorus by phytase are desirable traits in plant-growth promotion rhizobacteria (PGPR) particularly in Chilean Andisols which are characterized by low pH and high total P. However, little is known about the influence of soil properties that are specific to Andisol (low pH and metal toxicity) on the effectiveness of PGPR. Here, we assessed the effect of pH and metal cations on IAA and phytase activity of cell-associated proteins produced by two bacilli strains isolated from the rhizosphere of pasture plants. The production in vivo of IAA by Paenibacillus sp. SPT-03 was significantly increased (7-fold) when incubated in tenfold diluted culture medium, compared to the full-strength medium. At low pH (pH<5), phytase activity of cell-associated proteins and IAA production of Bacillus sp. MQH-19 was decreased, whereas they were increased in Paenibacillus sp. SPT-03. Moreover, phytase activity in vitro of cell-associated proteins and IAA production in both bacilli strains were significantly inhibited by 30-100% and 44-70% by concentrations of 10 mM and 350 μM Fe3+ and Al3+, respectively. At 350 μM Mn2+ IAA production was inhibited by 30-100% in both strains but there was no effect on phytase activity. This study shows that certain properties of Andisol may differentially affect some mechanisms related with PGPR efficiency.

Keywords: Andisol, Bacillus, indole acetic acid, phytase, rhizobacteria, rhizosphere.

1. Introduction

In the rhizosphere, defined as the soil influenced by roots, bacterial species that carry out functions which promote growth of plants have been defined as plant-growth promotion rhizobacteria (PGPR) (Martínez-Viveros et al., 2010). The enhancement of plant growth by members of bacilli strains, such as Bacillus and Paenibacillus, has been well documented (McSpadden, 2004; Ona, 2003). They promote plant growth by a number of mechanisms, including the solubilization of phosphorus and production of phytohormones, such as indole acetic acid (IAA) (Choudhary and Johri, 2009; Lal and Tabacchioni, 2009). Phytohormones such as IAA may indirectly improve P acquisition by plants by increasing root growth (Marschner et al., 2011). Moreover, Bacillus and Paenibacillus are also able to produce endospores which enhances their persistence and viability in soils (Lal and Tabacchioni, 2009; Nicholson, 2002). Hence, PGPR bacilli strains which have multiple mechanisms by which they promote plant growth have attracted considerable interest by microbiologists; biofertilizers containing Bacillus and Paenibacillus strains have been developed and commercialized. However, successful application of PGPR in the field is limited by a lack of knowledge on how environmental factors affect their survival and functionality in the plant rhizosphere.

Volcanic ash-derived soils (Andisols) in southern Chile have high contents of organic phosphorus (mainly as inositol phosphate, also known as phytate) (Borie and Rubio 2003). Rhizobacteria with the ability to mineralize phytate have been isolated and may improve P uptake by plants in these soils (Jorquera et al., 2008; Patel et al., 2010; Unno et al., 2005). However, these soils are also characterized by low pH (<5.5) as a consequence of natural acidification and long-term application of acidifying N fertilizers (particularly urea) (Mora et al., 2004). The low pH limits plant growth because the concentrations of metals (Al3+ and Mn2+) in the soil solution can reach toxic levels. It is known that soil pH and metal cations may affect many processes occurring in the rhizosphere (Greiner, 2004; Idris et al., 2007). Recently, Martinez et al. (2011) demonstrated that N fertilisation and factors present in Chilean Andisols (such as organic acids and metals) can have a relevant role in the occurrence and performance of culturable IAA-producing rhizobacteria. However, our knowledge if these factors also influence the effectiveness of soil inoculants in Chilean Andisols is still limited.

The main objective of this study was to assess the effect of pH and metal cations on the IAA and phytase activity produced by two bacilli strains isolated from the rhizosphere of pasture plants growing on an Andisol.

2. Material and methods

2.1. Bacilli strains

The bacilli strains used in this study were Bacillus sp. MQH-19 and Paenibacillus sp. SPT-03. Both strains were isolated from pasture containing graminaceous plants (Lolium sp., Festuca sp., Dactylis sp.) by Jorquera et al., (2011). Bacillus sp. MQH-19 was isolated from pasture located at the experimental station Maquehue (Andisol Freire series; 38°50' S, 72°41' W) of La Frontera University, which had a history of intensive annual fertilization with urea (300 kg ha-1), triple super phosphate (400 kg ha-1), potassium magnesium sulfate (300 kg ha-1), and calcium carbonate (500 kg ha-1). In contrast, Paenibacillus sp. SPT-03 was isolated from a natural pasture at San Pablo de Tregua (Andisol Liquine series; 39°36' S, 72°3' W), which had not received any chemical or manure inputs for 60 years. Our recent studies have demonstrated that both strains carry genes encoding β-propeller type phytases and have the ability to degrade phytate (Jorquera et al., 2011).

2.2. Biochemical and enzymatic characterization

Substrate utilization and enzyme release of Bacillus sp. MQH-19 and Paenibacillus sp. SPT-03 was evaluated by using commercial API® kits (20NE, 20E and ZYM; bioMerieux) which are commonly used for bacterial characterization. Bacilli strains were taken from overnight cultured in Luria-Bertani (LB) broth (g 1-1): 1 tryptone, 0.5 yeast extract, 0.5 NaC1 and pH 7.0. Cells were washed, suspended in sterile saline solution (0.9% w/v of NaC1) and then inoculated into each test tray following the procedure recommended by manufacturer.

The production of siderophores, iron (Fe) che-lating agents which may prevent the proliferation of plant pathogenic bacteria in the rhizosphere (Siddiqui, 2006), was evaluated on universal chrome azurol 'S' (CAS) medium as described by Alexander and Zuber-er (1991). After incubation for 4 days, the appearance of orange zones around the colonies was taken as an indicator of siderophore production.

To test the ability to release phosphate from insoluble inorganic and organic P forms, the strains were cultured under shaking (120 rpm) for 2 days at 30°C in NBRIP (National Botanical Research Institute's phosphate growth medium) (Nautiyal, 1999) and PSM (phytase-screening medium) (Kerovuo et al., 1998) broths, containing Ca-phosphate (Ca3(PO4)2) and Na-phytate (C6H18O24P6-xNa+-yH2O) as sole P sources, respectively. Un-inoculated broths served as controls. Phosphate released in liquid media was measured at 355 nm using the ammonium molybdate method (Heinonen and Lahti, 1981) and quantified by comparison with a standard curve prepared with known concentrations of PO42-.

2.3. Phytase activity in vitro

Bacillus sp. MQH-19 and Paenibacillus sp. SPT-03 were grown in 50 ml PSM broth (pH 7.0) for 2 days at 30°C and extracellular and intracellular phytase activity was measured in broth and protein extracts as follows. Bacterial cells and supernatant were separated by centrifugation (3,600 rpm for 5 min) and the supernatant was subjected to ammonium sulfate (0-85%) precipitation. The cell pellet was treated with lysozyme (5 mg ml-1) and sonicated (20 kHz constant frequency for 2 min) to induce cell lysis. The cell debris was centri-fuged (5,000 rpm for 5 min) and the supernatant was also subjected to ammonium sulfate precipitation. Both pellets were suspended and stored in Tris-Hl buffer pH 7.0 at -20°C. The phytase activity was assayed according to Greiner et al., (2004). Ten ul of crude protein extract was incubated with 270 μl of Na-phytate solution (2.5 mM of phytate in Tris-HCl buffer pH 7.0) for 30 min at 37°C. The reaction was stopped by addition of 1,150 μl of a 2:1:1mixture of fresh acetone, sulfuric acid, ammonium molybdate (10 mM) and 80 &#956;l of citric acid (1 M). Then the solution was centri-fuged (5,000 rpm for 5 min), the absorbance of the supernatant was measured at 355 nm and compared with standard curve of PO42-. One unit of phytase activity is equivalent to 1 μM P released in 1 min. Blanks were performed by adding the stop solution prior to substrate addition. Phytase activity under acidic condition (sodium acetate buffer pH 4.5) was also measured.

2.4. Production of IAA in vivo

The production of IAA by the bacilli strains was determined by colorimetric measurement at 530 nm using Salkowski's reagent as described by Patten and Glick (2002). Bacterial cells were grown under shaking (120 rpm) for 2 days at 30°C in LB broth and tenfold diluted LB broth (dLB) at pH 5.0 and pH 7.0 supplemented with tryptophan (1 mg ml-1) as IAA precursor. After incubation, the cells were centrifuged (3,000 rpm for 10 min at 4°C) and 1 ml of supernatant was combined with 2 ml of Salkowski's reagent (150 ml of 95-98% H2SO4, 7.5 ml of 0.5 M FeCL/6H2O, and 250 ml distilled water) and incubated for 30 min at room temperature. The quantification of IAA was carried out using a standard curve with known concentrations of pure IAA (Sigma-Aldrich, Co.).

Under acidic condition, the effect of several ami-no acids as precursors for production of IAA by bacilli strains was also assayed. Bacterial cultures in LB and dLB at pH 5.0 were supplemented with the following amino acids (1 mg ml-1): proline, phenylalanine, ala-nine, cysteine and methionine. Cultures supplemented with tryptophan (1 mg ml-1) were used as positive controls and uninoculated broths as negative controls. Tryptophan resulted in the highest IAA concentration and was therefore used for further assays.

2.5. Effect of pH and metal cations on phytase activity in vitro

Crude protein extracts from Bacillus sp. MQH-19 and Paenibacillus sp. SPT-03 grown in PSM were obtained as described above. The buffer used to determine the effect of pH on phytase activity of cell-associated proteins had the following composition: 100m M sodium acetate-acetic acid (pH 3.5, 4.5 and 5.5), 100 mM sodium acetate-HCI (pH 6.5), and 100 mM Tris-HCl (pH 7.0).

The in vitro effect of metal cations on phytase activity of cell-associated proteins was tested according to method described by Greiner et al., (2004). The total crude protein was incubated for 15 min at 37°C with Fe3+, Al2+ and Mn2+ at a final concentration of 10 mM at the optimal pH for cell-associated phytase activity in each strain: pH 7.0 for Bacillus sp. MQH-19 and pH 4.5 for Paenibacillus sp. SPT-03 .

2.6. Effect of pH and metal cations on production of IAA in vivo

To test the effect of pH, Bacillus sp. MQH-19 and Paenibacillus sp. SPT-03 were grown under shaking (120 rpm) for 2 days at 30°C in dLB supplemented with tryptophan at pH 5.0, 6.0 and 7.0.

To evaluate the in vivo the effect of metal cations on IAA production, bacilli strains were cultured for 2 days at 30°C in dLB (at pH 5.0) supplemented with the following metals (350 μM): Fe3+, Al2+ and Mn2+. This concentration represents the higher range of metal concentrations in the soil solution of Chilean Andisols (Mora et al., 2009; Rosas et al., 2007) Cell density (O.D. 600 nm) measurements showed that bacterial growth was not significantly affected by any treatment (data no shown). The IAA production was quantified as described above at point 2.4.

2.7. Analysis of data

Each experiment was performed in triplicate and repeated at least twice. Statistical analysis was performed by using the statistical software JMP®, version 5.0 (SAS Institute, Inc.). Before statistical analysis, the data were tested for normality. The significance of each treatment was established by one way ANOVA and the means were separated by Tukey's test (P≤ 0.05).

3. Results

3.1. Characterization of bacilli strains

The biochemical and enzymatic characterization of Paenibacillus sp. SPT-03 and Bacillus sp. MQH-19 is shown in the Table 1. Both strains showed common characteristics, such as production of α-glucosidase, β-glucosidase, acetoin, gelatinase, esterase, acid phosphatase and naphthol phosphohy-drolase; and utilization/assimilation of D-glucose, D-mannitol, D-mannose, N-acetyl-glucosamine, D-maltose, potassium gluconate, malate and citrate. Also, both strains were able to reduce nitrate and to produce siderophores on agar. In contrast to Bacillus sp. MQH-19, Paenibacillus sp. SPT-03 was able to produce β-galactosidase, ornithine decarboxylase and α-galactosidase; and utilize/assimilate rham-nose, D-melibiose and phenylacetic acids. Furthermore, Paenibacillus sp. SPT-03 produced all peptid hydrolases assayed (α-chymotrypsin, trypsin, valine arylamidase, cystine arylamidase and leucine aryl-amidase).

In relation to P release, Bacillus sp. MQH-19 had a higher phytate mineralization capacity, whereas Paenibacillus sp. SPT-03 showed a greater ability to solubilize inorganic P.

3.2. Phytase activity and production of IAA

The phytase activity assays showed that both bacilli strains had cell-associated phytase activity. After 30 min of incubation with phytate at pH 7.0, Bacillus sp. MQH-19 showed a mean value of 141 mU mg-1 protein, whereas no phytase activity was detectable in the crude protein extract from cells of Paenibacillus sp. SPT-03. However, at low pH (4.5), both Bacillus sp. MQH-19 and Paenibacillus sp. SPT-03 showed phytase activities with average values of 35 and 82 mU mg-1 protein, respectively. Phytase activity was not detected in the pellets of either bacilli strain.

Both bacteria were able to produce IAA when supplemented with tryptophan (Figure 1). However, IAA production was significantly higher (P≤0.05) in Paenibacillus sp. SPT-03 grown in dLB (32-37 μg; ml-1) compared with LB (4-6 μg ml-1), and compared with Bacillus sp. MQH-19 grown in dLB and LB (3-6 μg ml-1). Under acidic conditions (pH 5.0), IAA production was higher in LB and dLB supplemented with tryptophan compared to the other amino acids assayed, particularly in Paenibacillus sp. SPT-03 (Table 2).

3.3. Effect of pH and metal cations on phytase activity and production of IAA

The strains differed in optimal pH for phytase activity (Figure 2). Cell-associated phytase was highest at pH 7.0 for Bacillus sp. MQH-19 and at pH 4.5 for Paenibacillus sp. SPT-03. IAA production by Bacillus sp. MQH-19 was highest at pH 6.0 and decreased by 62% at pH 5.0 (Figure 2). For Paenibacillus sp. SPT-03, IAA production was highest at pH 5.0 and decreased by 42% at pH 7.0.

Ten mM Fe3+ and 350 ŞM Al3+ significantly decreased cell-associated phytase activity and IAA production (Figure 3). Compared to the controls, the cell-associated phytase activity of Bacillus sp. MQH-19 in full-strength LB was inhibited by 100 and 54% in the presence of Fe3+ and Al3+, respectively. Cell-associated phytase activity from Paenibacillus sp. SPT-03 was inhibited by 70 and 30% by Fe3+ and Al3+, respectively. The metals had similar negative effects on production of IAA in dLB (Figure 3). Compared to the control, presence of Fe3+ and Al3+ reduced IAA production in Bacillus sp. MQH-19 by 57-70% and in Paenibacillus sp. SPT-03 by >90%. In addition, 350 μM Mn2+ inhibited production of IAA by 44-50% in both bacilli strains, but did not affect the phytase activity of the crude protein extracts. Additionally, we carried out a chemical speciation analysis (GeoChemEZ) to evaluate the possible interaction of metals on phytase activity. The analysis revealed that 98% Al and 100% Fe form complexes with phytate while 32% of Mn could be found as Mn2+.

4. Discussion

Rhizobacteria can enhance plant growth by a number of mechanisms, hence it is likely that plant growth promotion by rhizobacteria is not the result of a single mechanism but rather the combined result of several mechanisms acting simultaneously (Martinez-Viveros et al., 2010). In the present study, we showed that Bacillus sp. MQH-19 and Paenibacillus sp. SPT-03 are able to produce diverse enzymes and utilize various substrates, and are able to utilize P from insoluble organic and inorganic P forms. Chilean Andisols are characterized by high concentrations of total and organic P (Borie and Rubio, 2003) and Pseudomonas, Enterobacter and Pan-toea with ability to mineralize/solubilize P and produce phosphorus hydrolases have previously isolated from pasture plants grown in Chilean Andisol (Jorquera et al., 2008). We have previously reported phytase activity in Bacillus sp. MQH-19 (1.9x10-10 kat mg-1) protein and Paenibacillus sp. SPT-03 (5.9x10-10 kat mg-1 protein) (Jorquera et al., 2011), but here we also show that our bacilli strains are capable to produce siderophores and synthesize IAA. Bacilli strains that can solubilize P and produce siderophore and IAA have widely been reported (Raddadi et al., 2008; Trivedi and Pandey, 2008). Previous studies have reported IAA productions between 12-48 μg ml-1 in Bacillus (Ali et al., 2009) and 4.6-44.6 μg ml-1 in Paenibacillus (Lebuhn et al., 1997). These values are similar to that observed for Paenibacillus sp. SPT-03 (4-37 μg IAA ml-1), but higher than that of Bacillus sp. MQH-19 (3-6 μg IAA ml-1). Bacilli strains that can solubilize P and produce IAA have been shown to promote the growth of maize and wheat (Beneduzi et al., 2008; Trivedi and Pandey, 2008)

The stimulation of IAA release by tryptophan found in the present study is in accordance with previous reports (Ali et al., 2009; Lebuhn et al., 1997). In Bacillus, the production of IAA has been described as tryptophan-dependent (Ali et al., 2009; Idris et al., 2007). However in the absence of tryptophan, the bacilli strains were also able to synthesize small amounts of IAA, suggesting that bacteria may use other aromatic amino acids as substrates for IAA production (Moat et al. 2002). Biosynthesis of IAA via tryptophan-independent pathways has been reported previously (Baca and Elmerich, 2007; Spaepen et al., 2006). In agreement with our results, IAA production by Pantoea agglomerans was lower in the presence of other aromatic amino acids than with trypto-phan (Sergeeva et al., 2007).

Interestingly, IAA production by Paenibacillus sp. SPT-03 was greater in the tenfold diluted LB broth than in the full-strength medium. It has been reported that IAA production by Azospirillum brasi-lense requires depletion of the carbon source in the growth medium (Ona et al., 2003) and is enhanced by N limitation (Malhotra and Srivastava, 2008).

The effect of pH and metals on phytase activity and IAA production differed between the Bacillus strains. Maximal phytase activity and IAA production were found at pH>5.0 in Bacillus sp. MQH-19 but at pH<5.0 in Paenibacillus sp. SPT-03. The studied Bacilli strains contain phytase-encoding genes with high similarity to p-propeller phytases of Bacillus (Jorquera et al., 2011). The optimal pH for β-propeller phytases is in the neutral and alkaline range (Fu et al., 2008). Hence the cell-associated proteins from Paenibacil-lus sp. SPT-03 seems to be an unusual β-propeller type phytase or contain another phytase types which we have not been identified, yet. However, a β-propeller phytase with maximal activity at acid pH has been reported for Bacillus licheniformis (Tye et al., 2002).

The effect of pH on IAA production is in agreement with studies with Azospirillum brasilense (Ona et al., 2003; Vande Broek et al., 2005). Maximal IAA production in A. brasilense was at pH 6.2 (Malhotra and Srivastava, 2008) and at pH 8 in Klebsiella pneu-moniae (Sachdev et al., 2009). In the present study, 10 mM Al3+ and Fe3+ inhibited phytate hydrolysis in both strains, whereas Mn2+ had no effect. Inhibition of phytate hydrolysis by Al3+ and Fe3+ but not by Mn2+ were reported by Greiner et al., (2004) in phytase from Pantoea agglomerans suggesting similarity between phytase from Pantoea and studied Bacilli strains. Nevertheless, the chemical speciation results indicated that metals might not be phytase inhibitors, but the hydrolysis was in fact affected by a phytate-ligand sequestration effect, which is in accordance to previous studies with fungal phytases (Dao, 2004)

The cations (Fe3+, Al+3 and Mn2+) inhibited IAA production, which is in agreement with Dimkpa et al., (2008), who demonstrated a negative effect of Fe3+ and Al+3 on auxin production by Streptomyces strains. Sid-erophores may reduce the toxic effect of metal cations by chelation (Dimkpa et al., 2008; 2009), but although the Bacillus strains can produce siderophores, this mechanism did not seem to be effective in our study.

5. Conclusions

Our study showed that environmental factors, including nutrient and amino acid availability, pH and metal cations, may influence the phytase activity and/ or IAA production by bacilli strains present in Chilean Andisols. Differences between two bacilli strains were observed, suggesting that Paenibacillus sp. SPT-03 may be more an efficient inoculant for Chilean Andi-sol, due to its higher phytase activity and IAA production under acidic condition. These differences between rhizobacteria should be taken into account to maximize the effectiveness of an inoculant when are applied in the different soil types, particularly in acidic soils.



This study was supported by FONDECYT No. 11080159, No.1100625 and International Cooperation CONICYT-BMBF cod. 2009-183. M.T. Fernandez acknowledges the UNU-BIOLAC Fellowship from United Nations University. O. Martinez-Viveros, D. Menezes-Blackburn and J. Acuna-Sobarzo thank the CONICYT and UFRO Ph.D. Scholarships.



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