Characterization of diazotrophic phosphate solubilizing bacteria as growth promoters of maize plants Caracterización de bacterias diazotróficas solublizadoras de fosfato como promotoras de crecimiento en plantas de maíz

Phosphorus is limiting for growth of maize plants, and because of that use of fertilizers like rock phosphate has been proposed. However, direct use of rock phosphate is not recommended because of its low availability, so it is necessary to improve it. In this study, a group of diazotrophic bacteria were evaluated as phosphate-solubilizing bacteria, for their production of indolic compounds and for their effects on growth of maize plants. Strains of the genera Azosporillum, Azotobacter, Rhizobium and Klebsiella, were quantitatively evaluated for solubilization of Ca3(PO4)2 and Rock Phosphate as a single source of phosphorous in SRS culture media. Additionally, the phosphatase enzyme activity was quantified at pH 5.0, 7.0 and 8.0 using p-nitrophenyl phosphate, and production of indolic compounds was determined by colorimetric quantification. The effect of inoculation of bacteria on maize was determined in a completely randomized greenhouse experiment where root and shoot dry weights and phosphorus content were assessed. Results showed that strain C50 produced 107.2 mg .L of available-P after 12 days of fermentation, and AC10 strain had the highest phosphatase activity at pH 8 with 12.7 mg of p-nitrophenol mL .h. All strains synthetized indolic compounds, and strain AV5 strain produced the most at 63.03 μg .mL. These diazotrophic bacteria increased plant biomass up to 39 % and accumulation of phosphorus by 10%. Hence, use of diazotrophic phosphate-solubilizing bacteria may represent an alternative technology for fertilization systems in maize


Introduction
Even though the phosphorus (P) content in soils is generally high, its availability for plants is often limited (Anwar and Jalaluddin, 1999), and therefore, P is considered the second most limiting element for crops, after nitrogen (Arcand and Schneider, 2006). Rock phosphate represents the greatest reservoir of P in nature; however, even the highest quality rock phosphate has low solubility and cannot always be recommended for direct use in crops. Traditional techniques to increase the quantity of soluble P in rock phosphate are not cost-effective, and therefore, new techniques are needed (Vanlauwea et al., 2000). Richardson et al. (2009) suggested improve the efficiency of phosphorus fertilizers by using a specific inoculant capable of improving availability of P in soil, or assimilation of this element by the roots. The phosphate solubilizer bacteria can increase the P availability in soils through different mechanisms as the organic acid excretion, the phosphatase enzymes activity or the synthesis of chelating agents (Rodríguez and Fraga, 1999). Although immediate effects of phosphate rock used in maize crops are frequently not seen (Vanlauwea et al., 2000), it has been reported that bacterial strains of the genera Serratia and Pseudomonas capable of solubilizing rock phosphate can promote plant growth of maize by improving the uptake of P by plants when they are fertilized with phosphate rock as source of P (Hameeda et al., 2008). Microorganisms belonging to the genera Pseudomonas, Bacillus, Rhizobium, Azotobacter and Azospirillum, among others, frequently have the ability to solubilize phosphorus (Rodríguez and Fraga, 1999). This microorganisms are capable to synthetize plant growth regulators as indolic compounds (Patten and Glick, 2002;Cassán et al., 2009), defined as a group of organic substances with an important role in cell division, elongation, differentiation, development and growth of roots, tropism regulation and adult plant structure (Woodward and Bartel, 2005). The main indolic compound produced by bacteria is indol acetic acid (IAA), an active biological form of the auxins, which stimulates radical system growth (Dobbelaere et al., 2003;Vessey, 2003) and increases the uptake of the nutrients by the plant. The aim of this study was to present the role of diazotrophic bacteria of the genera Azospirillum, Azotobacter, Bradyrhizobium, Rhizobium and Klebsiella as phosphate solubilizing bacteria and indolic compounds producers, as well as to determine in a preliminary way the effect of the inoculation of maize plants with these bacteria and the assimilation of phosphorus under greenhouse conditions.
Phosphorus solubilization assay. Determination of released P by the evaluated strains was carried out in SRS culture broth (Sundara and Sinha, 1963;Nautiyal, 1999) and SRS broth supplemented with rock phosphate (RP) named SRS-RP, in all cases all the assays were realized in triplicate. Inocula were produced in sterile solution of NaCl (0.85 % w/v) at OD600 = 0.500 (approximately 10 8 cells .mL -1 ). One mL of each inoculum was inoculated in 24 mL of the culture broth SRS and SRS-RP in flasks (capacity of 125 mL). These broths were incubated at 30 ± 2 °C, 150 rpm (Labline Hertz 3525). At the end of this period, the soluble P was quantified using the phosphomolybdenum blue method using an absorbance of 712 nm (Murphy and Riley, 1962;Watanabe and Olsen, 1965). The final pH was evaluated by a potentiometer (Consort C861). Rock Phosphate. The rock phosphate was from a deposit of the Pesca municipality (Boyacá -Colombia) with the following chemical composition: 30 % P2O5, 40 % Ca, 12 % Si, 0.1 % Mg, 40 ppm Mn, 30 ppm Cu, 10 ppm Mo, 300 ppm Zn and 3 % of moisture.
Determination of phosphatase enzymes. Quantification of phosphatase enzyme activity was estimated at de 12 días de fermentación y que la cepa AC10 tuvo la más alta actividad fosfatasa a pH 8 con 12.7 mg de p-nitrofenol mL .h -1 . Todas las cepas sintetizaron compuestos indólicos y la cepa AV5 produjo la más alta cantidad con 63.03 µg .mL -1 . Estas bacterias diazotróficas incrementaron la biomasa de las plantas por encima del 39 % y de la acumulación de fósforo por el 10 %. Aquí, el uso de bacterias diazotróficas solubilizadoras de fosfato puede representar una alternativa tecnológica para los sistemas de fertilización en plantas de maíz. were randomized with 10 repetitions per treatment under semi-controlled conditions in greenhouse with a maximum temperature of 33.14 °C and a minimum pH 5.0, 7.0 and 8.0. The inoculum was produced in SRS culture broth without P, and it was inoculated at 1 % of the effective volume (EV) and incubated in agitation at 120 rpm, 30 ± 2 °C for 48 -96 h according to the growth kinetics of each strain during her stationary phase (data not shown). In order to evaluate the activity of phosphatase enzymes it was used p-nitrophenyl phosphate as substrate in all cases; to quantify the acid phosphatases it was used an acid buffer (sodium acetate 0.5 M and MgCl2 0.1 M) and for alkaline phosphatases it was used an alkaline buffer (Tris-Cl 0.5 M and MgCl2 0.1 M) (Patel et al., 2010). The reaction was incubated at 35 °C (ShelLab-WGM) for 1 hour and then NaOH 20 mM was added to stop the reaction. The released concentration of p-nitrophenol was measured by spectrophotometry at 450 nm (Tabatabai and Bremmer, 1969).

Production of indolic compounds.
Cultures were grown at 30 ± 2 °C in K-lactate culture medium supplemented at 1% with L-triptophane (10 %) for 48 to 96 h (based on the growth kinetics of each strain) and were centrifuged at 10000 rpm for 10 minutes. Indolic compounds were determined in the supernatant by Salkowsky's method at 540 nm (Carreño et al., 2000;Glickmann and Dessaux, 1995). fructose and glucose, strains of A. brasilense and A. lipoferum produced gluconic acid that is involved in phosphate solubilization process.
The solubilization capacity of the two sources of P by the strain BRCG3 (Klebsiella variicola) exceeded 50 mg .L -1 , at evaluated conditions, confirming that the genus is able to solubilize insoluble sources of P. Ahmad et al., (2008) reported the genus Klebsiella as organic acid producer from the glucose metabolism to solubilize phosphates present in the soil solution, Ahemad and Saghir (2011) reported levels of solubilization up to 294 mg .L -1 using as source of P tricalcium phosphate.
Both strains A. chroococcum AC1 and A. vinelandii AV5 released similar amount of P, however, the values were significantly lower in comparison with the ones obtained from Rhizobium sp. C50, meanwhile the strain A. chroococcum AC10 showed similar quantities of soluble P in the culture medium in comparison with this strain. Several studies have reported that the genus Azotobacter does not present high levels of solubilization. In that way, Kumar and Narula (1999) found values of P solubilization between 0.18 and 0.19 mg .L -1 for native strains of Azotobacter chroococcum using rock phosphate as source of P. Similarly, Husen (2003) reported that A. vinelandii Mac 259, was not able to solubilize tricalcium phosphate in Pikovskaya culture medium. Those results do not coincide with our results reported here especially with A. chroococcum AC10, which presented one of the greatest activities.
Phosphatase enzymes determination. All tested strains produced phosphatases at the pH values tested (figure 1). Strains SP7, C15, C16, USDA110 and BRCG3 had the lowest enzymatic activity at the three pH values. In contrast, AC1, AC10 and AV5 (genus Azotobacter) and C50 showed the highest values of enzymatic activity at the pH values evaluated. Strain AC10 showed the greatest activity at pH 8 with 12.70 mg of p-nitrophenol mL -1 .h -1 , at pH 7.0 AV5 demonstrated the greatest enzymatic activity with 8.77 mg of p-nitrophenol mL -1 .h -1 . At pH 5.0 AC1 presented an enzymatic activity of 9.01 mg of p-nitrophenol mL -1 .h -1 . Overall, the results show that the strains of genus Azotobacter have the greatest phosphatase activity in the different pH evaluated in comparison with the other strains.
Characterization of the bacteria was complemented with the phosphatase activity measurement, which has not been studied in the genera of the present study at great length before. Other authors have reported productions of phosphatases from 2.62 µg mL -1 .h -1 released p-nitrophenol by Pantoea ananatis to 70.98 temperature of 12.85 °C during the assay. Plants were watered once every three days. Agronomic variables (shoot and root length and dry weigh) were evaluated 20 days after inoculation. Shoot P content was determined for each treatment (Bray and Kurtz, 1945).
Statistical analysis. Data were analyzed by SPSS version 17.0. One-way analyses of variance (ANOVA) and comparison among treatments were done by Tukey's HSD. All analyses were performed at the P = 0.05 level.

Results and discussion
Phosphorus solubilization. The results of phosphorus solubilization capacity of the diazotrophic bacteria evaluated are shown in table 1. All the strains solubilized tricalcium phosphate and phosphate rock in broth.
In the presence of tricalcium phosphate, the concentration of soluble P was between 12.12 and 107.23 mg .L -1 after 12 days of fermentation. Strain C50 (Rhizobium sp.) produced the highest concentration with a final pH value of 4.02. In contrast, in SRS broth supplemented with phosphate rock, the concentration of soluble P varied between 3.22 and 62.78 mg .L -1 . Strain AC10 had the greatest solubilization of P with a final pH value of 5.25. The phosphorus solubilization in culture media supplemented with tricalcium phosphate and phosphate rock was accompanied by decreases in the initial pH (7.2) due to the activity of the different bacteria, ranged between 4.02 and 6.92. In a generally way, a negative correlation was observed between the available P and the pH values, thus the greatest solubilization by the evaluated bacteria was presented at lower pH values.
The potential of genus Rhizobium as a phosphate solubilizing bacterium has been previously described using sources as hydroxyapatite, FePO4, AlPO4 and Ca3(PO4)2 (Sridevi and Mallaiah, 2009). They used tricalcium phosphate as source of P and reported solubilization levels between 156 and 620 mg .L -1 of P2O5, meanwhile in the present study it was shown an solubilization of 107.23 mg .L -1 . B. japonicum, has been reported as a non-phosphate solubilizing bacteria (Fernandez et al., 2005). However, our results showed that the strain USDA110 was able to solubilize a great quantity of P using the two sources of phosphates.
None of the strains of the genus Azospirillum was able to solubilize high concentrations of P in comparison with the other evaluated strains. Ramachandran et al., (2007) and Vikram et al., (2007) reported similar results for several species of Azospirillum. Nevertheless Rodríguez et al., (2004) reported that in presence of released p-nitrophenol (µg mL -1 .h -1 ) by Burkholderia cepacia, either because of acid or alkaline phosphatase activity (Oliveira et al., 2009), thus the obtained results from this study are among the mineralization rates reported by other bacteria genera.
The evaluated strains in the present study, were isolated from different soils, crops and climatic conditions therefore it is likely that several factors, as temperature, pH and redox potential, could affect the phosphatase enzyme expression (Šarapatka, 2003). Additionally, the enzyme activity was evaluated during the stationary phase of each strain. In this phase the organic P associated to death cell, could act as an inducer of phosphatase synthesis (Jagadish et al., 2001). All of the tested strains expressed phosphatase enzyme at all three tested pH levels. However, according with Šarapatka (2003) acid phosphatases are more common than alkaline phosphatases in soil microorganisms, which may account for the large production of phosphatases in acid environments such as in tropical soils. In contrast, in this study the greatest expression of phosphatases occurred under alkaline conditions. Indolic compounds production. All of the tested strains synthetized indolic compounds from tryptophan as  the precursor (figure 2). Strain AV5 had the greatest production of indolic compound with an average of 63.03 µg .mL -1 , followed by AC10 with 54.41 µg .mL -1 .
Previous studies have reported that the genus Rhizobium synthetizes indol acetic acid from several tryptophan isomers (DL and L) (Perrine et al., 2004) and reaches production levels up to 90.6 µg .mL -1 with tryptophan at 1% (Datta and Basu, 1997) and 267.5 µg .mL -1 with tryptophane at 4 % (De and Basu, 1996). In the present study, the production of indolic compounds with tryptophane at 0.1 % by Rhizobium sp. C50, was lower that the reported in literature, however, this could be related with the concentration of tryptophane in the culture medium (Datta and Basu, 1997).
For genus Azotobacter it has been reported several species that can produce different quantities of IAA. A. chroococcum produced 12.2 µg .mL -1 of IAA, A. beijerinckii produced 12 µg .mL -1 of IAA, A. vinelandii produced between 11 µg .mL -1 and 49.07 µg .mL -1 of IAA (Fiorelli et al., 1996;Ravikumar et al., 2004). These results are overcome by the results obtained in this study with the different species of Azotobacter evaluated. Similarly, the production of indolic compounds by the strain B. japonicum USDA110 were superior that those found in literature where are reported productions of 11.8 µg .mL -1 of IAA (Badawi et al., 2011) and 6.62 µg .mL -1 of IAA in absence of tryptophan (Cassán et al., 2009), in this way in the present study tit was evaluated the production of indolic compounds in presence of tryptophan as precursor and it could stimulate the production.
The genus Klebsiella has been reported as indolic compound producer before. Ahemad and Saghir (2011) found a production of de 42 µg .mL -1 of IAA, this result can be compared with the levels of production obtained from the strain BRCG3, under the established conditions of this work.
It is known that in the genus Azospirillum the primary pathway for production of IAA is the indol-3-piruvic acid pathway (Patten and Glick, 1996;Malhotra and Srivastava, 2008). This pathway is dependent on tryptophan (Malhotra and Srivastava, 2008). Although tryptophan was used in the current study as a precursor, the concentration of indolic compounds produced by the strains of the genus Azospirillum was lower than the concentration produced by the other strains with the exception of the strain SP7. Also it is known that Azospirillum synthetizes these compounds in the absence of tryptophan, as reported by Cassán et al. (2009) who reported that Azospirillum brasilense produced 13.16 µg .mL -1 of IAA, which is consistent with our results.
Inoculation test under greenhouse conditions. Maize plants inoculated with strains AC1, AC10, USDA110 and BRCG3, had the highest production of biomass compared to the chemical control with Hoagland's solution supplemented with rock phosphate (HS*RP) (table 2). Inoculation with strain BRCG3 increased root dry weight by 39 % greater than the control HS+PR, with a mean of 1.01 g, followed by treatment with strain AC10 with 29 % of increase and 0.87 g (table 2). The strains AC1 and AC10 increased shoot dry weight in approximately 33 % in comparison with the control (table 2).
The plants inoculated with strains AC1 and BRCG had the greatest concentration of phosphorus in shoot with Table 2. Shoot and root dry weight and phosphorus concentration in shoot of maize plants under greenhouse conditions. For each variable, data with the same letter are not significantly different at a confidence level of 95%. The results of the growth promotion tests are similar to the results obtained by Hameeda et al. (2008) where it was observed under greenhouse conditions a significant increase of the dry weight of maize plants due to inoculation with foreign strains that showed the ability to solubilize phosphates and other mechanisms to promote plant growth. This shows that isolations from other crops, or even from out of rhizosphere, can promote plant growth. In the present study it was evaluated strains that were not isolated from maize, which showed an important effect on maize growth promotion.

Treatments
It was observed a major increment in maize root dry weight in comparison to shoot dry weight. Some plant species spent a great portion of their total dry matter in roots growth when are farmed in P deficiency (Hill et al., 2006). Liu et al., (2004) showed that in two different genotypes of maize, roots respond first than shoot in P deficiency through the production of lateral roots and radicular hairs because of the plant need to cover a greatest area for searching this element. In the present study, the P was supplied as non-available phosphate rock. The inoculation with phosphate solubilizing bacteria could have a positive effect over the acquisition of P by the plant and over plant development.
The growth-promoting effect was evident by A. chroococcum strains AC1 and AC10, which are capable to solubilize phosphate and produce indolic compounds. It has been reported the effect of strains of A. chroococcum, phosphate solubilizing and phyto-hormone producer bacteria, over plant growth parameters on several wheat varieties. It has been found that phosphate solubilization and indolic compounds production have a positive influence in plant height (19 %), crop yield (14 %) and root biomass (12 %), with reports were the lower doses of fertilization are matched or overcome (Kundu and Gaur, 1980;Kumar et al., 2001).
In the study by Kumar and Narula (1999), increased plant growth was attributed to the production of plant growth promoting substances and to the phosphate solubilizing activity of A. chroococcum. This bacterial species can improve the availability of P in soil, in that way; according to Chabot et al., (1998) phosphate solubilization is an effective mechanism in plant growth promotion.
B. japonicum USDA110 also showed important results in maize growth promotion. Previously it has been de-monstrated that strains of Bradyrhizobium, under greenhouse conditions, improved germination of up to 8 % and stimulated shoot weight by 35 % and root weight by 32 % (Cassán et al., 2009). Other authors report increases in soy up to 31% (Molla et al., 2001) beans up to 64 % (Gupta et al., 1998) and peanuts up to 37 % (Badawi et al., 2011). In the present work, the strain USDA110 increased shoot dry weight in maize plant up to 20 % and root dry weight up to 23 %.
The results of assay control were higher than those of treatment inoculated with Rhizobium sp. C50. This contrast with the reports of strains of R. leguminosarum, that after 20 days  and Rhizobium etli, that after 40 days (Gutiérrez and Martínez, 2001), have the ability to significantly increase dry weight compared with controls .
With Klebsiella, Farzana et al., (2009) found an increase of roots dry weight and volume in potato plants compared with the non-inoculated control, and concluded that this effect may be the result of bacterial indolic compounds production. These results agree with the ones of the present study where it is shown that the strain that belongs to this genus present the higher values in root dry weight (1.1 g) regarding the non-inoculated control (0.61 g).
It is well known that root tissues are extremely sensitive to changes in concentrations of indolic compounds, like IAA, and also, that the root development could be affected by production of IAA by plant growth-promoting rhizobacteria (Tanimoto, 2005). The synthesis of indolic compounds by the evaluated microorganisms could stimulate the root system by the development of lateral roots and apical divisions of the meristem that conduces to the roots growth (Patten and Glick, 1996;Vessey, 2003;Dobbelaere et al., 2003), and also to the increase of the plant access to soil's nutrients, allowing a greatest production of vegetal biomass (Patten and Glick, 1996;Vessey 2003;Barazani and Friedman, 1999). The evaluated strains in the present study showed an in vitro ability to synthetize indolic compounds using tryptophan as precursor and they could use this amino acid found in root exudates free in rhizophere (Dakora and Philips, 2002;Malhotra and Srivastava, 2009;Barea et al., 1976).
The ability of bacterial genus to increase the P supply to the plant using rock phosphate has been well documented, Yu et al., (2012) found that in walnut trees, Pseudomonas chlororaphis and Arthrobacter pascens with the ability to solubilize P under in vitro conditions, increases the shoot and root dry weights with significant differences up to 22 % when were compared with the non-inoculated control and phosphate rock added. In addition, the authors found an increase in the concentration of P up to 21 % compared with the control. According with Kumar et al., (2001) can be suggested that the inoculation with bacteria capable to solubilize phosphates, in this case phosphate rock, increases the availability of this element in substrate, and as consequence, increases its acquisition by the plant. The description above coincides with the results of the present study where was shown that the use of phosphate rock as source of P in maize crops with the inoculation of bacterial strains can increase up to 10 % the uptake of this element in comparison with the non-inoculated control supplied with phosphate rock.

Conclusion
In the present study the results allow to affirm in a preliminary way that the evaluated diazotrophic bacteria can increase the maize plant biomass in shoot and root, and also, the accumulation of P in plant. This represents a possible alternative for the maize phosphate fertilization system in our country with sources of low solubilization like as rock phosphate.