PHOSPHATE-SOLUBILIZING BACTERIA ASSOCIATED WITH RUNNER BEAN RHIZOSPHERE

Soil microorganisms, especially rhizobacteria, play a key role in soil phosphorus (P) dynamics and the subsequent availability of phosphate to plants. Utilization of phosphate-solubilizing bacteria as biofertilizers instead of synthetic chemicals is known to improve plant growth through the supply of plant nutrients, and may help to sustain environmental health and soil productivity. The main purpose of this study was to identify new phosphate-solubilizing bacteria isolated from runner bean rhizosphere. Ten out of 25 isolated bacterial strains solubilized Ca3(PO4)2 in qualitative and quantitative P-solubilization. The strain that exhibited the highest potential to solubilize Ca3(PO4)2, was selected for further determination of the mechanisms involved in the process. The medium pH was measured, organic acids released in the culture medium were identified by HPLC analysis, and the acid and alkaline phosphatase activities were determined. Our results showed that strain R7 solubilized phosphorous through the production of various organic acids such as lactic, isocitric, tartaric and pyruvic acids, and that it can be used as a potential biofertilizer.


INTRODUCTION
The runner bean (Phaseolus coccineus L.) is a climbing perennial vegetable often grown as an annual crop for dry seeds or immature pod production; it is also grown as an ornamental vine.Although of minor importance in the United States, the crop is of importance in some parts of Europe.In the United Kingdom, for instance, runner bean is frequently grown in order to produce a reliable crop of green beans for commercialization.In Romania the lack of scientific knowledge about the biology and ecology of runner bean has contributed to the "slower" progress of this species (Munteanu et al., 2007).
Phosphorus is one of the major nutrients for biological growth and development, but for plants is also a limiting one, due to its low availability of soluble forms in soil (Vessey, 2003).The biggest reserves of P in soil are in mineral forms such as apatite, hydroxyapatite and oxyapatite and in organic forms such as inositol phosphate, phosphomonoesters, phosphodiesters and phosphotriesters, which cannot be directly assimilated by plants (Rodriguez et al., 1999, Gamalero et al., 2011).
Solubilization of insoluble and fixed forms of P is an important issue for increasing soil P availability to plants (Nautiyal, 1999).Microorganisms are responsible for this process, in particular bacteria, which constitute up to 50% of the soil population capable of P solubilization (Khan et al., 2009).A high proportion of phosphate-solubilizing microorganisms (PSM) belongs to the rhizosphere where they are metabolically more active compared to other sources due to the release of root exudates (Vazquez et al., 2000).
The mechanisms by which PSM can solubilize inorganic P are not fully understood, but the production of organic acids seems to be a major mechanism (Alam et al., 2002).The most common organic acids produced by PSM are gluconic, 2-ketogluconic, malonic, oxalic, succinic and fumaric acids (Rodriguez et al., 1999).Other mechanisms that have been involved in the solubilization of inorganic phosphate are the release of H + , the production of chelating substances and inorganic acids, and the synthesis of exopolysaccharides (Gamalero et al., 2011).On the other hand, phosphatase activity may participate in inorganic phosphate solubilization (Park et al., 2011).
Large reserves of P are found in most agricultural soils due to the regular application of chemical fertilizers (Rodriguez et al., 1999).However, 75-90% of added P fertilizers is rapidly precipitated, soon after application, by Fe, Al and Ca complexes present in the soil, making it unavailable to plants (Gyaneshwar et al., 2002).Excessive use of chemicals in traditional cultivation practices has led to a reduction in soil fertility by modifying its physical, chemical and biological characteristics, also causing environmental degradation.As an eco-friendly alternative, P biofertilizers in the form of rhizobacteria can help in increasing the availability of accumulated phosphates for plant growth by solubilization (Gyaneshwar et al., 2002).Therefore, the main purpose of this study was to identify new phosphate-solubilizing bacteria isolated from runner bean rhizosphere and to determine their potential to be used as biofertilizer in organic agriculture.In addition, there are no data concerning the phosphate solubilization processes in runner bean rhizosphere and the mechanisms involved in bacterial phosphate solubilization are very controversial.Therefore, a second objective of our study was to identify the main mechanism used by a runner bean rhizobacteria strain to solubilize inorganic phosphate.

Isolation of bacteria
Bacteria strains were isolated from the rhizosphere of a field-grown runner bean crop from the Experimental Farm, University of Agriculture Sciences and Veterinary Medicine, Iasi County, Romania, using the serial dilution method (Dunca et al., 2004).Aliquots (0.1 ml) were plated on Bunt and Rovira nutrient medium and incubated at 28°C for seven days.Isolates were re-streaked on the same nutrient medium, checked for purity and stored on slants at 4°C.

Plate assay
The ability of rhizobacterial strain to solubilize P was assessed following the plate method (Chaiharn et al., 2011) using Pikovskaya (PVK) agar medium containing Ca 3 (PO 4 ) 2 as insoluble inorganic form of P. Bacterial strains were streaked in the center of PVK agar plate and incubated at 28°C for seven days.Bacterial colonies surrounded by a halo, indicating phosphate removal, were visually observed and measured.The solubilization index was measured according to the formula: the sum of colony diameter and the halo zone diameter/the colony diameter (Alam et al., 2002).Three replicate plates were used for each isolate.

Quantitative P assay
Bacteria strains were grown in triplicate in 25 ml Pikovskaya (PVK) agar medium with and without Ca 3 (PO 4 ) 2 on a gyratory shaker (190 rpm) at 28°C (Nautiyal, 1999).Control flasks were not inoculated with bacteria.A 5-ml aliquot was aseptically removed from each flask at 0, 3, 5 and 7 days, centrifuged at 4 500 rpm for 30 min and assessed for pH.A supernatant volume of 1.5 ml from the flasks with Ca 3 (PO 4 ) 2 was further centrifuged at 14 000 rpm for 30 min and the amount of P released was estimated using the phosphomolybdate method (Artenie et al., 2008).

PCR amplification, 16S rDNA sequence analysis and bacterial identification
Total genomic DNA was extracted and purified according to (Ausubel et al., 2002).The 16S rDNA fragment was amplified with the primers F27 (AGAGTTTGATC-MTGGCTCAG) and R1492 (TACGGYTACCTTGT-TACGACTT) (Muyzer et al., 1993).PCR reaction was performed in 20 µl of reaction mixture containing 5 µl PfuDNA Polymerase 10X Buffer with MgSO 4 (Promega), 1µl dNTP mix (10mM each), 10 pmol of forward and reverse primers and 1U PfuDNA Polymerase (Promega).The reaction conditions included an initial denaturation of 2 min at 95°C followed by 30 cycles of 30 s at 94°C, 30 s at 52°C and 2 min at 72°C.The amplification fragment was purified from gel with Wizard® SV Gel and PCR Clean-up System (Promega Inc, Madison, WI, USA) and then sequenced in forward and reverse reactions, using the Dye Terminator Cycle Sequencing (DTCS) Quick Start Kit (Beckman Coulter).The sequencing cycle consisted of 30 cycles at 96°C for 20 s, 52°C for 20 s and 60°C for 4 min.The sequencing product was purified by ethanol precipitation according to the manufacturer's instructions, and sequence analysis was performed using the CEQ8000 Investigator (Beckman CoulterTM) software.The 16S rDNA sequence was analyzed using BLAST algorithm against the SILVA database (Pruesse et al., 2012).

Production of organic acids during phosphate solubilization
For organic acid identification and quantification, 1 ml of culture from each flask was centrifuged at 14 000 rpm for 30 min and filtered through a 0.2-μm PES syringe filter (Roth, Germany); 20-μl of culturefiltrate was injected on a PRP-x300 PSDVB-Sulfonic acid column (Hamilton, Germany) and organic acids were monitored using a Bischoff Lambda 1010 UV detector at 220 nm.The mobile phase consisted of 1 mN sulfuric acid with a flow rate of 1 ml/min (Walser, 1988).

Phosphatase activity assay
Acid and alkaline phosphatase activities were determined using a modified assay of Juma and Tabatabai (De Freitas et al., 1997).One milliliter of culture supernatant was incubated at 28°C with 1 ml 25mM p-nitrophenyl phosphate and 4 ml modified universal buffer, pH 6.5 or pH 11 (Thornton et al., 1975), for assay of acid or alkaline phosphatase.After 3 h of incubation, the reaction was terminated by adding 1 ml 0.5 M CaCl 2 and 4 ml 0.5 M NaOH.The assay mixtures were centrifuged for 10 min and the yellow color measured at 410 nm using a Beckman Coulter DU 720 spectrophotometer.

Statistical analysis
The experimental data were statistically processed using ANOVA two-factor with replication and Stu-

Qualitative screening of bacterial strains isolated from runner bean rhizosphere
Ten out twenty-five isolated bacterial strains showed clearly visible halos around their colonies on PVK agar medium after 7 days of incubation.The solubilization index, based on colony diameter and halo zone, ranged from 0.33 to 3.19 (Table 1).The highest solubilization index was recorded for R4 (3.19) followed by R1 (2.89) and R2 (2.87).

Quantitative estimation of phosphate solubilization in PVK broth
Phosphate solubilization results recorded in liquid media showed that all the isolates have the potential to solubilize the inorganic form of P as indicated by a gradual increase in the amount of soluble P in the medium.The bacterial strain that exhibited the highest Psolubilizing potential was R7 (19.5 μg P/ml), identified by 16S DNA analysis as belonging to the Pseudomonas genus.The strain was used for further tests.
The pH value in the uninoculated control flasks remained almost the same (Fig. 1).A gradual pH decrease from the initial value of 7.03 to 4.92 on the 7th day was noticed in PVK broth supplemented with tricalcium phosphate (Fig. 1).Moreover, it was observed that the amount of solubilized P increased along with the medium pH decrease.The lowest pH value was assessed on the 7th day (4.92) when the amount of solubilized P reached the maximum value (19.5 μg P/ml).

HPLC analysis
HPLC analysis of the culture filtrates was performed in order to identify the organic acids produced by R7 in the presence or absence of inorganic phosphate source from the PVK medium.The growth of the R7 strain on the PVK medium supplemented with tricalcium phosphate during the 7-day incubation resulted in the accumulation of a major organic acid, namely lactic acid, compared to the control.Other organic acids accumulated during incubation were isocitric, tartaric and pyruvic acids.The organic acids accumulated in PVK supplemented with Ca 3 (PO 4 ) 2 on day 3 were isocitric and lactic (Fig. 2).Day-5 chromatographic analysis showed an important accumulation of lactic and tartaric acids compared to the previous day when an increase of solubilized P was also observed (Fig. 2).On day 7, when the amount of solubilized P was maximum, the major acid accumulated in PVK supplemented with Ca 3 (PO 4 ) 2 was lactic acid compared to the control, followed by tartaric and pyruvic acids (Fig. 2).

Phosphatase activity
The acid phosphatase activity varied from 1.37 pNP/ml/h to 4.30 pNP/ml/h in the case of R7 strain cultivated in PVK without Ca 3 (PO 4 ) 2 , and between 2.15 and 7.40 pNP/ml/h for the culture from PVK supplemented with the calcium phosphate.The alkaline phosphatase activity recorded in PVK medium without Ca 3 (PO 4 ) 2 varied between 1.59 and 7.76 pNP/ml/h, and from 3.33 to 7.75 pNP/ml/h for PVK supplemented with Ca 3 (PO 4 ) 2 .The analysis showed that there are no significant differences between the acid and alkaline phosphatase activity recorded in PVK medium compared to PVK supplemented with the inorganic P source.

DISCUSSION
Bacteria isolated from runner bean rhizosphere were first assayed for their ability to solubilize insoluble phosphate using a plate screening method.The production of clearing zones around the microbial colonies on PVK medium containing tricalcium phosphate as the sole P source indicated the presence of phosphate-solubilizing traits.The solubilization index results were similar with those recorded by Alam et al. (2002) for phosphate-solubilizing bacteria (PBS) isolated from maize.
Because of the contradictory results between plate halo detection and phosphate solubilization in liquid media (Fankem et al., 2006), bacterial strains exhibiting a clear halo on PVK plates were tested for P solubilization in PVK broth.The results showed that the best strain that solubilized the same phosphate source in liquid media was one of the strains that did not have the greatest solubilization index.This confirms the fact that the plate method is reli-able only for isolation and preliminary characterization of phosphate-solubilizing microorganisms The tricalcium phosphate solubilization during the 7 days of incubation was accompanied by a gradual pH decrease.The decrease in pH value is in agreement with the findings of Sharma et al. (2012) and Chaiharn et al. (2011), who observed that the solubility of Ca-phosphate increased exponentially with pH decrease.The medium pH drop indicates the production of organic acids as suggested by Mehta et al. (2001).The production of organic acids seems to be the main mechanism used by rhizobacteria for mineral phosphate solubilization (De Freitas et al., 1997, Rodriguez et al., 1999).The organic acid produced by phosphate-solubilizing bacteria most frequently is gluconic acid (Rodriguez et al., 1999, Vyas et al., 2009, Gulati et al., 2010).Other major organic acids that have been identified among phosphate solubilizers in high amounts are oxalic and citric acid (Alam et al., 2002;Rashid et al., 2004), 2-ketogluconic acid (Muleta et al., 2013) and succinic acid (Panhwar et al., 2012).Organic acids found in small quantities during phosphate solubilization are lactic, formic (Vyas et al., 2009), malic, propionic (Panhwar et al., 2012), acetic and fumaric (Alam et al., 2002).However, our results indicate that lactic, isocitric, tartaric and pyruvic acids are the main acids produced by the strain R7 (isolated from runner bean rhizosphere).Moreover, it has been found that during the 7 days of incuba-tion the amount of solubilized P gradually increased with the accumulation of lactic acid in combination with isocitric, tartaric and pyruvic acids.This indicates that organic acid production may have a central role in the solubilization of an insoluble phosphate source.
Several authors suggested that phosphatase activity underlies the mechanism of mineral phosphate solubilization (Tarafdar et al., 1987;Ponmurugan et al., 2006;Pantujit et al., 2010;Park et al., 2011).Our results indicate that there is no significant positive correlation between the amount of solubilized P and acid and alkaline phosphatase activity (Fig. 3).This can be explained by the fact that acid and alkaline phosphatases catalyze the hydrolysis of organic P compounds such as esters and anhydrides of orthophosphoric acid, and do not solubilize rock phosphates (De Freitas et al., 1997).However, the synthesis of these phosphatases is enhanced when the level of inorganic P in the growth medium is limiting (Kaleeswari, 2007;Nannipieri et al., 2011).Hence, an apparent relationship between P solubilization and phosphatase activity can be considered coincidental.

CONCLUSION
Our results confirm the potential use of strain R7, a rhizospheric isolate from runner bean roots, for the development of a biofertilizer formula based on phosphorous solubilizing bacteria for organic agriculture.The main mechanism used by R7 to solubilize inorganic P consisted in lowering the medium pH due to the production of organic acids such as lactic and tartaric acids.The relationship between phosphatase activity and phosphate solubilization can be considered coincidental.

Fig. 1 .
Fig. 1.Changes in P solubilization (left) and pH value (right) recorded for strain R7 during 7 days of incubation in PVK broth supplemented with Ca 3 (PO 4 ) 2 .
dent t-test to calculate the differences between the different solubilization indices and alkaline phos-phates activities; Pearson correlation was used for exploring the relation between solubilized P and phosphatase activity.

Table 1 .
Solubilization index of rhizobacteria isolates after 7 days of incubation The values are means ± SEM