BMC Microbiology

Research article

BioMed Central

Open Access

Organic acid production in vitro and plant growth promotion in maize under controlled environment by phosphate-solubilizing fluorescent Pseudomonas
Pratibha Vyas and Arvind Gulati*
Address: Plant Pathology and Microbiology Laboratory, Institute of Himalayan Bioresource Technology (CSIR), P.O. Box No. 6, Palampur (H.P.)176 061, India Email: Pratibha Vyas - vyasp2000@yahoo.co.in; Arvind Gulati* - gal_arvind@yahoo.co.in * Corresponding author

Published: 22 August 2009 BMC Microbiology 2009, 9:174 doi:10.1186/1471-2180-9-174

Received: 16 April 2009 Accepted: 22 August 2009

This article is available from: http://www.biomedcentral.com/1471-2180/9/174 © 2009 Vyas and Gulati; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: Phosphorus deficiency is a major constraint to crop production due to rapid binding of the applied phosphorus into fixed forms not available to the plants. Microbial solubilization of inorganic phosphates has been attributed mainly to the production of organic acids. Phosphatesolubilizing microorganisms enhance plant growth under conditions of poor phosphorus availability by solubilizing insoluble phosphates in the soil. This paper describes the production of organic acids during inorganic phosphate solubilization and influence on plant growth as a function of phosphate solubilization by fluorescent Pseudomonas. Results: Nineteen phosphate-solubilizing fluorescent Pseudomonas strains of P. fluorescens, P. poae, P. trivialis, and Pseudomonas spp. produced gluconic acid, oxalic acid, 2-ketogluconic acid, lactic acid, succinic acid, formic acid, citric acid and malic acid in the culture filtrates during the solubilization of tricalcium phosphate, Mussoorie rock phosphate, Udaipur rock phosphate and North Carolina rock phosphate. The strains differed quantitatively and qualitatively in the production of organic acids during solubilization of phosphate substrates. Cluster analysis based on organic acid profiling revealed inter-species and intra-species variation in organic acids produced by Pseudomonas strains. The phosphate-solubilizing bacterial treatments P. trivialis BIHB 745, P. trivialis BIHB 747, Pseudomonas sp. BIHB 756 and P. poae BIHB 808 resulted in significantly higher or statistically at par growth and total N, P and K content over single super phosphate treatment in maize. These treatments also significantly affected pH, organic matter, and N, P, and K content of the soil. Conclusion: The results implied that organic acid production by Pseudomonas strains is independent of their genetic relatedness and each strain has its own ability of producing organic acids during the solubilization of inorganic phosphates. Significant difference in plant growth promotion by efficient phosphate-solubilizing Pseudomonas strains point at the need for selecting potential strains in plant growth promotion experiments in conjunction with various phosphate substrates for their targeted application as bioinoculants.

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2-ketogluconic acid (Sigma.BMC Microbiology 2009. high alkalinity. and passed through 0. Plant growth under controlled environment Seeds of Zea mays var. and washed thrice with sterile distilled water were germinated at 25°C in moist sterile vermiculite. Several Pseudomonas species have been reported among the most efficient phosphate-solublizing bacteria and as important bio-inoculants due to their multiple biofertilizing activities of improving soil nutrient status. formic acid. Burkholderia.000 rpm for 10 min. Previously we have reported the isolation. selection.5% tricalcium phosphate (TCP). oxalic acid. Methods Bacterial strains Nineteen phosphate-solubilizing fluorescent Pseudomonas included in the present studies were isolated from the rhizosphere of Hippophae rhamnoides growing in the cold deserts of Lahaul and Spiti in the trans-Himalayas and characterized based on their phenotypic characters and 16S rDNA gene sequencing [8. The nutritional problems in such soils are often specific in respect of the low phosphorus availability resulting from their high phosphorus-fixing capacity due to high calcium content [10].85% NaCl (OD600 = 1. malonic acid. Quantitative estimation of P-liberated from inorganic phosphates was done using vanado-molybdate method as described earlier [8]. Mussoorie rock phosphate (MRP).9]. secretion of plant growth regulators.biomedcentral. and characterization of stress-tolerant and efficient phosphate-solubilizing fluorescent Pseudomonas from the cold deserts of the Himalayas [8. Phosphorus release from insoluble phosphates reported for several soil microorganisms has been attributed mainly to the production of organic acids and their chelation capacity [1-3]. 9:174 http://www. and Lichrosphere RP-18 column 250 mm × 4.7-9].5 1. Time (min) 0–8 8–14 14–25 Flow rate (ml/min) 0. succinic acid. Germany). The values were presented as the mean of three replicates.2 Page 2 of 15 (page number not for citation purposes) . Udaipur rock phosphate (URP) and North Carolina rock phosphate (NCRP) at 28°C for 5 days at 180 rpm in a refrigerated incubator shaker (Innova Model 4230. Eluates were detected at λ 210 nm and identified by retention time and co-chromatography by spiking the sample with the authentic organic acids. and lactic acid. Production of organic acids during phosphate solubilization The bacterial strains grown in triplicate in 10 ml NBRIP broth supplemented with 0. New Brunswick Scientific. Waters 717 plus autosampler. Improving soil fertility by releasing bound phosphorus by microbial inoculants is an important aspect for increasing crop yield. The bacterial strains were maintained at -70°C in nutrient broth supplemented with 20% (v/v) glycerol.com/1471-2180/9/174 Background Phosphorus (P) is an essential macronutrient often limiting the plant growth due to its low solubility and fixation in the soil. Bacillus. and Agrobacterium [5. The aim of the present study was to explicate organic acid production during solubilization of inorganic phosphates and effect on plant growth as a function of phosphate solubilization by fluorescent Pseudomonas. Erwinia.6]. The mobile phase was 0. Waters 600 controller. and low organic matter content. propionic acid and tartaric acid (Supelco. Increased solubilization of fixed soil phosphates and applied phosphates ensuring higher crop yields has been reported on inoculation of phosphate-solubilizing bacteria including Pseudomonas. Waters™ pump. Inoculum preparation The bacterial strains grown in 20 ml trypticase soya broth (TSB) for 48 h at 28°C were centrifuged at 10. Girija surface sterilized with 20% sodium hypochlorite for 3 min. Micrococcus.0). The organic acids were quantified by reference to the peak areas obtained for the authentic standards for gluconic acid (Sigma-Aldrich. Soils in the cold deserts of Lahaul and Spiti in the Indian trans-Himalayan region latitude 31° 44' 57" and 32° 59' 57" North and 76° 46' 29" and 78° 41' 34" East are often with low moisture status.6 mm and 5 μm particle size (Merck. USA). malic acid. and suppression of soil-borne pathogens [5.1% ortho-phosphoric acid (Merck.000 rpm for 10 min. Rhizobium.4]. Uniformly germinated seeds were coated with the water slurry of charcoal-based microbial Table 1: HPLC elution-profile program. Flavobacterium. Germany) in the gradient of flow rate as given in Table 1. Achromobacter. The vast potential of microorganisms for improving productivity in the region remains unexploited [11].9]. and the pellets suspended in 0. Each replicate was analyzed in a single run on HPLC for 76 samples for the four phosphate substrates. citric acid. Waters inline degasser AF.22 μm nylon filter.4 0. Direct periplasmic oxidation of glucose to gluconic acid is considered as the metabolic basis of inorganic phosphate solubilization by many Gram-negative bacteria as a competitive strategy to transform the readily available carbon sources into less readily utilizable products by other microorganisms [1. Detection and quantification of organic acids was done on Waters 996 High Performance Liquid Chromatogram (HPLC) equipped with PDA detector. USA) were centrifuged at 10. USA). USA). The bacterial suspensions were separately mixed with sterilized activated charcoal (4:6 v/w) to give a CFU of approximately 107/g of charcoal-based bacterial inoculants.

The influence of PSB treatments on plant growth and soil properties was evaluated in comparison to the uninoculated control treatments with or without TCP and SSP. two P.1%.5. strains. formic acid by three P. P. fluorescens. All strains exhibited the production of malic acid excepting one Pseudomonas sp. During MRP solubilization the production of oxalic and gluconic acid was also detected for all the strains (Table 4). respectively. ten Pseudomonas trivialis strains. The pots were placed in a complete randomized block design with four replications under 550 μM photon m-2 s-1 mixed incandescent and fluorescent illumination. trivialis strains. strain. The production of other organic acids was restricted to some strains: 2-ketogluconic acid to three Pseudomonas spp. K. Page 3 of 15 (page number not for citation purposes) . fluorescens and four Pseudomonas spp. strains in combined application with NPK with TCP as the phosphate source. trivialis. The production of lactic acid was restricted to one strain of both P. and muriate of potash @ 80 kg K/ha. total and available K by flame photometric method.. P. URP and NCRP by Pseudomonas fluorescens strain. The plants were removed carefully under a gentle flow of tap water after 90 days of sowing.96. strains. Chemical analyses The soil samples were air dried and sieved for determining pH. fluorescens and Pseudomonas sp. trivialis. Estimation of available P was estimated by sodium biocarbonate method [14] and available N by alkaline permanganate method [15].biomedcentral. During URP solubilization the production of oxalic and gluconic acid was detected for all the strains (Table 3). The germinated seeds treated with the water slurry of sterilized activated charcoal without inoculum were used for the control treatments. strains. strain no other strain showed oxalic acid production. P. three P. trivialis strains and one strain each of P. three Pseudomonas poae strains. one P. succinic acid to one strain each of P. 5 × 105 CFU/seed) and two seeds per pot sown in 15 cm diameter pots filled with 2 kg non-sterilized sandy-loam soil. P and K.03%. During TCP solubilization all strains showed the production of gluconic and 2-ketogluconic acids (Table 2). fluorescens strain. Experimental design and data analyses Randomized block design with two factor factorial arrangement was adopted for conducting the experiments. available N. strains. lactic acid by five P. strains. Organic matter was determined by the modified Walkley and Black method [12]. None of the strains showed citric acid production during URP solubilization. poae strains. P was applied @ 120 kg P/ha either as single super phosphate (SSP) or tricalcium phosphate (TCP) according to the various treatments. available N 0. fluorescens and two Pseudomonas spp. trivialis.0011%. The production of other organic acids was limited to some strains: 2-ketogluconic acid to five P. P. fluorescens strain. and citric acid to three P. MRP. USA. two P. lactic acid to five P. trivialis. malic acid by two P.BMC Microbiology 2009. The samples were oven-dried at 70°C for 3 days to a constant weight for determining the dry weight. strain and succinic acid excluding one Pseudomonas sp. trivialis. trivialis. The production of 2-ketogluconic acid was shown by one Pseudomonas poae. trivialis and four Pseudomonas spp. The soil used had pH 6. strains and one strain each of P. formic acid to two P. The phosphate-solubilizing bacterial (PSB) treatments included one P. strain.01 using SPSS Software version 7. available P 0. P. strains. lactic acid to three P. and citric acid by one Pseudomonas sp.013% and available Ca 0.028%. 1). The data were subjected to one-way analysis of variance (ANOVA) and the mean of treatments compared by Duncan's Multiple Range Test at p ≤ 0. poae and four Pseudomonas spp. poae and P. trivialis and three Pseudomonas spp. strains. 16/8 h light/dark cycle and 50– 60% RH at 25 ± 2°C in an Environment Control Chamber. 9:174 http://www. strains. poae. fluorescens and four Pseudomonas spp. and malic acid to four P. plant height (aerial parts). Data on root length. and available Ca in ammonium-acetate extracts [13]. and five Pseudomonas spp. N and K were applied in the form of ammonium sulfate @ 240 kg N/ha. total P by vanado-molybdate yellow colour method. fluorescens and two Pseudomonas spp. trivialis strains. Ca and organic matter content. P. The plant samples were oven-dried and powdered for estimation of total N. Results Production of organic acids HPLC analysis of the culture filtrates was done to identify and quantity the organic acids produced during the solubilization of TCP. ten P. one P. strains. formic acid by P. trivialis. 2004). poae. fluorescens and three Pseudomonas spp. P. and P. Apart from one Pseudomonas sp. trivialis and Pseudomonas sp. root dry weight and shoot dry weight were recorded.. Cluster analysis based on the organic acid profiles was performed using STATISTICA data analysis software system version 7 (StatSoft® Inc. Estimation of total N was done by modified Kjeldahl's method. formic acid to six P. TCP was chosen as phosphate substrate since P-deficiency in soils of the cold deserts of Lahaul and Spiti is attributed mainly to the presence of insoluble diand tricalcium phosphates. poae and Pseudomonas sp. available K 0.. organic matter 3. and five Pseudomonas spp. trivialis. P. In NCRP solubilization the production of oxalic acid and gluconic acid was detected for all the strains (Table 5). trivialis strains. poae and three Pseudomonas spp. Tulsa. fluorescens and three P. strains. succinic acid by three Pseudomonas spp.com/1471-2180/9/174 inoculants (approx. strains (Fig.

BMC Microbiology 2009. CA = citric acid and PA = propionic acid. MalA = malonic acid. 9:174 http://www. Udaipur phosphate supernatant rock spiked phosphate of Pseudomonas with OA (c). for HPLC chromatograms of authentic organic acids (a) and culture supernatant of Pseudomonas trivialis strain BIHB 747 grown for 5 days at 28°C in NBRIP broth with tricalcium phosphate (b).organic and tricalcium North acids phosphate Carolina (a) and culture rock (b). 2-KGA = 2-ketogluconic acid. MA = malic acid. GA = gluconic acid. Mussoorie rock phosphate (d). OA = oxalic acid. Udaipur rock phosphate (c). SA = succinic acid. and North Carolina rock phosphate spiked with OA (f). (f) Mussoorie trivialis strain rock BIHB phosphate 747 grown (d). TA = tartaric acid. LA = lactic acid.com/1471-2180/9/174 North Figure HPLC 5 days chromatograms at Carolina 1 28°C in rock NBRIP phosphate of broth authentic with (e). FA = formic acid.biomedcentral. North Carolina rock phosphate (e). Page 4 of 15 (page number not for citation purposes) .

7 1965.0 ± 3.7 ± 0.0 987.7 ± 5.1 2551.7 BIHB 763 718.0 ± 4.9 177.7 ± 3.3 ± 2.9 4114.7 32676.3 ± 1.4 ± 1.4 ± 4.5 ± 2.1 ± 2.3 ± 4.8 667.3 ± 3.4 779.8 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 53.7 20607.52 3.3 ± 2.7 ± 5.0 ± 2.9 321.3 ± 9.7 18336.0 ± 2.1 BIHB 804 805.0 ± 4.6 ND ND ND ND 7.7 ± 3.7 ± 2.7 BIHB 749 801. BMC Microbiology 2009.3 536.0 ± 3.3 175.1 ND ND ND ND ND ND ND ND 44.7 ± 1.8 BIHB 747 743.2 30. 9:174 (page number not for citation purposes) .1 475.3 ± 3.7 ± 6.4 ± 3.3 ± 4.9 173.8 ND ND 103.9 911.9 2656.6 20090.6 17819.2 1236.3 ± 7.3 172.4 175.5 ± 3.5 18800.5 ± 1.8 ND 240.0 ± 3.0 ± 3.3 17038.0 ± 2.82 3.7 ± 2.0 ± 5. Organic acid (μg/ml) Strain P-liberated (μg/ml) Final pH Oxalic Gluconic 2-KGA Lactic Succinic Formic Citric Malic Total organic acids (μg/ml) P.8 23.1 49.7 ± 4.7 ± 2.0 ± 2.2 17929.8 1654.7 ± 3.0 ± 6.0 93.7 ± 4.8 ND 65.9 19340.1 BIHB 750 774.2 2012.0 19611.3 ND ND 117.5 17901.0 ± 1.4 18840.9 BIHB 757 775.6 179.3 ± 4.0 583.0 ± 4.5 ± 2.7 19722.3 ± 3.7 ± 2.2 96.7 ND ND ND 114.0 659.3 ± 1.2 ± 2.3 ± 4.0 18054.1 21412.6 ± 1.3 ± 8.0 20547.6 154.7 21666.9 193.7 ± 2.5 116.9 ± 2.3 ± 2.0 ± 3.0 ± 1.6 25.5 195.63 3. 2-KGA = 2-ketogluconic acid.4 323135.5 ND 209.8 BIHB 752 805.5 BIHB 769 806.0 2051.8 216.3 ± 5.9 BIHB 813 631.8 18035.0 ± 3.8 21961.0 ± 5.90 3.3 378.9 46.Page 5 of 15 http://www.7 ± 5.20 3.42 3.0 ± 4.0 ± 3.7 ± 4.3 ± 0.3 ± 4.4 373228.0 ND ND ND ND 129.92 3.5 ± 2.5 17937.2 176. fluorescens BIHB 740 768.7 772.5 ± 1.3 ± 3.0 ± 3.3 19934.4 532.5 22739.7 224.3 ND 96.2 106.4 BIHB 745 827.7 18350.2 22467.6 Pseudomonas spp.53 3.00 3.0 BIHB 756 802.0 19858.0 ± 1.93 ND ND ND ND ND ND ND ND ND ND ND ND ND ND 7.3 257.8 1172.3 ± 1.7 ± 3.3 ± 2.3 BIHB 759 751.3 P. BIHB 751 318.4 1307.9 330.0 ± 3.5 75.8 17464.0 ± 4.7 BIHB 808 821.7 ± 6.7 ± 2.0 ± 5.3 ± 6.50 3.0 ± 3. poae BIHB 730 768.6 20730.0 ± 2.2 1042.6 210.1 18216.2 BIHB 811 717.8 ± 3.5 12024.0 ± 3.97 4.6 122.1 1718.58 3.2 BIHB 736 778.4 3128.7 ± 4.1 217. trivialis BIHB 728 771.0 ± 5.3 ± 1.3 ± 3.0 22233.5 251.0 ± 4.6 163.0 ± 5.3 ± 5.3 ± 2.1 21726.65 3.9 509.1 14427.98 3.3 1961.8 ± 1.7 ± 3.4 19797.4 ± 2.4 928.3 ± 2.0 ± 2.0 ± 6.9 4215.3 18057.70 3.3 ± 7.0 ± 5.4 ± 1.2 ND ND ND 28.biomedcentral.7 132.3 ± 4.1 207.55 3.8 1181.7 P.3 21496.2 18624.5 ± 2.1 2249.40 3.4 14.7 ± 3.7 Values are the mean of three replicates ± standard error of the mean.0 ± 3.72 4.1 14665.6 3178.6 797.0 ± 2.7 ± 3.8 ND 913.5 Total organic acids (μg/ml) 3.2 1520.0 ± 6.com/1471-2180/9/174 Table 2: Organic acid production by fluorescent Pseudomonas during tricalcium phosphate solubilization.5 17745.0 ± 2.3 ± 1.8 ± 5. ND = not detected.

40 6.70 3.01 5.62 3.9 10.1 5055. 9:174 (page number not for citation purposes) .9 8190.7 5250.0 ± 1.10 7.0 ± 3.0 ± 3.7 ± 6.5 ± 2.90 9.03 4.3 ± 3.8 7616.3 5097.1 ± 4.0 ± 3.5 57.1 ± 0.1 ± 2.6 8289.Page 6 of 15 http://www.3 ± 1.5 11169.6 ND ND ND 102.3 12.0 ± 2. fluorescens BIHB 740 Pseudomonas spp. poae BIHB 730 BIHB 752 BIHB 808 P.4 631.6 2964.2 ND ND 67.3 ± 5.7 ND ND ND ND ND ND ND ND ND 20.8 ND ND ND 164.8 7126.1 11.4 ± 0.4 5061.2 9.10 8.7 ± 2.6 ± 0.78 3.1 ± 0.biomedcentral.7 ± 1.5 ND ND ND 108.3 ± 7.0 7119.5 ND 599 ND ND ND ND ND ND ND ND ND 56.3 ND 5753.0 ± 5.5 658.3 ± 1.6 5983.7 ± 0.5 11.8 ± 0.5 ND 138.4 ± 1.0 ± 2.7 ± 1.4 ± 0.30 3.4 ± 0.5 ND ND 103.5 ± 2.2 97392.20 3.0 ND ND ND ND ND ND ND ND ND ND 128.6 ± 2.0 ± 0.5 ± 1.9 7496.8 11.1 ± 3.1 ± 0.0 ± 6.6 255.8 4412.50 5.3 ND 4350.1 6850.3 16.7 ± 5. 2-KGA = 2-ketogluconic acid.3 383.8 ± 1.6 3528.5 7662.8 ± 0.9 ND 549.60 3.01 6.79 3.75 4.0 26.70 3.7 ± 9.8 8.1 ± 0.5 235.3 ± 1.0 ± 5.5 10. trivialis BIHB 728 BIHB 736 BIHB 745 BIHB 747 BIHB 749 BIHB 750 BIHB 757 BIHB 759 BIHB 763 BIHB 769 P.8 6962.05 3.3 ± 0.71 3.7 6859.3 ± 3.0 ± 0.6 ± 3.5 1117.1 ± 1. ND = not detected.00 4.4 ± 0.1 51.04 7.3 ± 5.3 ± 0.7 ± 6.7 ND ND 34.0 ± 3.6 ± 0. Organic acid (μg/ml) Strain P-liberated (μg/ml) Final pH Oxalic Gluconic 2-KGA Lactic Succinic Formic Citric Malic Total organic acids (μg/ml) P.0 ± 3.3 ND ND 41.78 3.1 ND ND ND ND ND 93.3 ± 2.7 Total organic acids (μg/ml) Values are the mean of three replicates ± standard error of the mean.7 9. BIHB 751 BIHB 756 BIHB 804 BIHB 811 BIHB 813 8.7 104925.78 3.7 12.7 41.8 9.8 ± 2.6 5467.5 ± 0.3 ± 3.4 7540.8 ± 0.0 13.4 5839.4 8.0 5936.1 1463.7 ± 2.4 10903.0 ± 5.6 5183.3 ± 1.03 9.4 ND ND 35.7 ± 1.9 ± 0.3 ± 0.6 ± 1.87 3.8 12.4 ± 3.4 52.7 ± 4.4 36.0 7116.1 ND 266.05 3.0 ± 0.7 ± 5.4 7162.0 41.5 ± 1.2 ± 2.9 17.4 ± 0.1 ND 35.83 4.05 1.04 5.2 12.8 5455.9 ± 0.9 6743.0 121.3 ± 1.6 7013.7 ± 0.2 5971.0 ± 3.90 3.5 ND ND ND 42.6 ± 0. BMC Microbiology 2009.3 ± 5.9 7974.09 7.4 ± 1.6 6676.72 3.05 5.7 ± 6.0 ± 2.04 14.11 4.0 5459.2 ± 0.com/1471-2180/9/174 Table 3: Organic acid production by fluorescent Pseudomonas during Udaipur rock phosphate solubilization.5 ± 3.32 14.6 ND ND 43.5 25.02 7.7 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0 ND ND 53.8 ± 1.0 7921.6 ± 0.0 92.30 4.

2 ± 0.2 14.0 ± 2.3 ND ND 930.0 ± 5.3 ± 4.8 9460.7 ± 3.03 BIHB 813 2.4 ± 0.9 234.7 ± 1.0 370.4 15.2 BIHB 763 12.0 ± 6.62 3.2 12042.4 7871.7 8503.0 ± 0.52 3.biomedcentral.04 BIHB 759 11.6 P.0 ± 4.4 ± 2.0 ± 5.8 8504.8 ± 2.00 4.0 ± 0.7 ± 1.0 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 142.1 ± 2.3 ± 6.8 ± 2.9 ± 0.1 18. fluorescens BIHB 740 3.4 8997.5 ± 2.4 ± 4. ND = Not detected.1 BIHB 745 5.52 3.0 ND ND ND ND 114.0 ± 5.55 4.89 3.0 ± 0.0 8830.3 BIHB 736 13.3 ± 2.4 ND ND 468.06 BIHB 752 6.Page 7 of 15 http://www.9 ± 0.4 P.0 ± 8.2 16039.3 ± 7.7 264.0 ± 3.3 ± 2.8 ± 0.7 ± 4.52 3.8 ± 0.7 ± 1.2 10007.1 50. 2-KGA = 2-ketogluconic acid.0 ± 4.4 ± 2.2 10586.50 3.0 ± 7.63 3.0 ± 0.9 Values are the mean of three replicates ± standard error of the mean.5 ND 349.0 ± 5.2 ± 0.0 ± 3.0 ± 5.4 ± 0.3 10016.53 3.7 ND 183.6 ± 1.1 ± 0.7 ± 5.9 44.9 13444.7 136.7 11.0 ± 8.5 11633.2 ND ND 32.6 34.05 15.8 10396.0 ± 0.7 204347.0 9510.3 ± 1.5 13854.6 ± 0.3 ± 2.9 2802.0 ± 5.1 Pseudomonas spp.0 ND ND ND ND ND ND ND ND ND ND 19.5 42.6 ± 1.5 16.0 ± 3.7 ND ND 36.6 9120.2 39.1 ND ND ND ND ND 293.1 ± 2.5 ± 2.2 ± 4.2 BIHB 749 8.2 153.59 3. trivialis BIHB 728 11.4 10742.1 ± 1.0 ± 6.90 3.1 627.9 10422.1 ± 0.4 ± 0.3 BIHB 747 12.com/1471-2180/9/174 Table 4: Organic acid production by fluorescent Pseudomonas during Mussoorie rock phosphate solubilization.5 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 22.0 13910.4 16.0 ± 4.9 36.67 3.0 16.0 ND ND 39.0 ± 1.9 13749. Organic acid (μg/ml) Strain P-liberated (μg/ml) Final pH Oxalic Gluconic 2-KGA Lactic Succinic Formic Citric Malic Total organic acids (μg/ml) P.9 ± 1.8 ± 0.0 ± 3.9 11.7 15.4 BIHB 757 9.0 ± 4.0 ND 1019.4 13549.3 ± 0.7 ND ND ND ND 87.3 9329.8 ± 2.2 ± 0.6 ± 2.7 12.3 BIHB 811 2.3 ± 1.1 126.8 ND 4574.0 9314.53 5.6 ± 1.1 ± 0.49 3. BMC Microbiology 2009.62 3.2 ND 22.7 8460.6 13765.0 ± 4.3 19. poae BIHB 730 4.7 ± 0.0 ± 0.4 8051.02 BIHB 769 6. 9:174 (page number not for citation purposes) .8 39.7 ND ND 84.3 9693.7 ± 3.0 ± 0.7 ± 2.6 7941.7 ± 0.5 ± 1.6 37.1 15.6 197042.7 14.7 ± 0.03 BIHB 808 8.3 ± 0.1 70.9 ND ND ND ND ND 356.65 4.04 BIHB 750 4.1 BIHB 756 12.9 10.8 10140.4 Total organic acids (μg/ml) 3.0 ± 0.4 BIHB 804 8.7 334.5 15727.9 10016.4 12027.3 18.3 40.7 9128.3 ± 7.3 ND 40.4 14. BIHB 751 2.63 3.9 ± 1.9 11690.4 9394.1 ND ND ND ND ND 39.1 7076.4 14.0 ± 3.8 ± 1.8 8443.

5 4640.9 4762. trivialis BIHB 728 191.2 ± 0.2 ± 0.5 ± 1.9 42.6 ND 121.2 Total organic acids (μg/ml) 3.72 3.3 ± 2.1 ± 0.8 10.1 ND 10.2 ND ND ND 75.7 ± 0.7 4649.6 ± 2.2 ± 2.3 ± 1.8 ± 0.0 ± 0.2 BIHB 769 224.48 3.0 ± 6.9 4724.07 14.9 ND 173.0 ND ND 10.90 3.6 BIHB 750 145.1 ± 1.0 ± 7.8 ND ND ND ND ND ND ND ND ND ND ND ND 45.8 ND ND ND ND ND ND ND ND ND ND ND ND ND 59.3 10.6 Values are the mean of three replicates ± standard error of the mean.8 ± 1.1 BIHB 752 204.8 11.7 ± 5.3 BIHB 745 168.3 3241.3 ± 3.1 ± 2.3 ± 0.0 22. 9:174 (page number not for citation purposes) .8 ND ND 13.81 3.6 BIHB 763 161.3 ± 1.0 ± 5.0 ± 5.0 4405.0 4384.2 ± 1.1 ± 1.3 2549.4 3880.73 3.7 ± 1.8 ± 2.0 ± 5.4 BIHB 749 177.5 ± 2.3 4458.0 ± 0.3 ± 1.1 4442.9 ND 10.78 3. fluorescens BIHB 740 236.7 ± 0.3 ± 7.3 ± 1.3 ± 2.0 ± 5.3 ± 6.0 ± 3.1 ± 0.6 4975.6 4448.3 ND ND ND ND ND ND ND ND ND ND ND 26.9 4633.8 3780.3 11.0 17.6 15. Organic acid (μg/ml) Strain P-liberated (μg/ml) Final pH Oxalic Gluconic 2-KGA Lactic Succinic Formic Citric Malic Total organic acids (μg/ml) P.0 299.88 3.1 3810.72 3.0 ND ND ND 29.82 3.3 ± 0. BMC Microbiology 2009.6 Pseudomonas spp.3 ± 0.8 31.6 13.80 3.4 11.4 5057.3 46.5 11.92 4.3 ± 1.1 ± 1.2 ND ND ND 55.3 ± 2.7 ND ND ND ND ND ND ND ND 104.1 ± 4.5 ± 1.8 BIHB 804 161.55 3.6 4675.1 11. ND = Not detected.5 ± 1.4 BIHB 747 173.0 ± 4.0 32.1 4362.2 230.1 BIHB 813 92.7 ± 1.92 3.3 ± 5.5 ± 0.7 ± 1.0 ± 1.9 ± 1.0 ± 4.6 ND 931 3834.7 ± 2.3 BIHB 759 178.0 ± 0.2 265.7 ± 1.7 BIHB 808 193.7 5014.4 BIHB 756 164.7 ± 3.0 ± 4.6 6102.0 ± 6.0 10.6 ± 1.Page 8 of 15 http://www.0 ± 4.8 ± 1.5 2991.2 ± 0.biomedcentral.1 9.8 8.8 ± 0.4 ND 42.6 9.3 10.2 12.1 3901.0 ± 2.0 BIHB 811 173.0 38. BIHB 751 123.2 9.9 5342.2 ND ND ND ND ND ND ND ND ND ND 41.7 P.73 3.0 54.0 ND 10. 2-KGA = 2-ketogluconic acid.8 ± 3.8 ± 0.6 4672.2 6035.5 ± 1.3 ± 2.8 ± 1.3 3770.7 4395.5 ND ND ND ND ND 59.2 BIHB 757 175.0 ± 6.6 4681.com/1471-2180/9/174 Table 5: Organic acid production by fluorescent Pseudomonas during North Carolina rock phosphate solubilization.4 4947.9 ± 0.5 4542.2 15.0 85881.65 3.0 ± 0.1 4996.6 3687.0 ± 1.6 ND ND ND ND 113.0 ± 7.0 415.5 5331.6 10.0 ± 1.5 84010.3 ± 2.3 ± 0.7 ± 1.0 BIHB 736 172.5 16.0 ND 33.1 ± 4.9 10.0 4420.9 4760.89 3.7 P.7 ± 1.4 ± 0.0 ± 1.81 3.4 39.2 ND 40.2 4587. poae BIHB 730 163.70 3.7 ± 4.0 ± 0.

respectively. 631. Formic acid production only by P.0 μg/ml formic acid.3 μg/ml oxalic acid. 16. P. fluorescens and three Pseudomonas spp. 4. 25. Oxalic acid production detected only for Pseudomonas sp.3–532. trivialis. Lactic acid production shown only by P.1–108 μg/ml succinic acid.1 and 93. of phosphate-solubilizing (c) Mussoorie rockfluorescent phosphate.7–19340 μg/ml gluconic acid. formic acid to P. 23. BIHB 804 was 53. 5).4–468 Page 9 of 15 (page number not for citation purposes) .. 96–2249 μg/ml succinic acid. citric acid to one strain each of P.6 μg/ml oxalic acid. and (d) North Carolina rock phosphate after 5 days incubation at 28°C.3–15727 μg/ml gluconic acid. BIHB 751 was 318.BMC Microbiology 2009. 9:174 http://www. poae and Pseudomonas sp.biomedcentral. 7076.3 μg/ml.3–164 μg/ml lactic acid..4–255 μg/ml 2-ketogluconic acid. 14.com/1471-2180/9/174 Dendrogram tricalcium 5 Figure days incubation 2 phosphate. trivialis BIHB 745 and P. based aton 28°C (b) organic Udaipur acid rock profiles phosphate. trivialis BIHB 728 and Pseudomonas sp. and 75–4215 μg/ml malic acid.3 μg/ml 2-ketogluconic acid. strains. 18. (b) Udaipur rock phosphate.6 μg/ml.5–4350 μg/ml malic acid. fluorescens strain. trivialis BIHB 763 was 35. poae. Quantitative difference in the production of organic acids was observed during the solubilization of phosphate substrates by Pseudomonas strains (Tables 2.6–39. respectively. Organic acid production during URP solubilization varied from 8–26. (c) Mussoorie rock phosphate.2 μg/ml citric acid.7 μg/ml during TCP solubilization. succinic acid to one strain each of P. During MRP solubilization the quantities of organic acids estimated in the culture filtrates were 10. 3. P.7– 10903 μg/ml gluconic acid.8–132.7 and 49. 41. and 34. Pseudomonas and (d) North grown Carolina in NBRIP rock phosphate broth with after (a) Dendrogram based on organic acid profiles of phosphate-solubilizing fluorescent Pseudomonas grown in NBRIP broth with (a) tricalcium phosphate. 56. and malic acid to one P.5–65. The quantities of organic acids produced during TCP solubilization ranged from 216. fluorescens and Pseudomonas sp.

98a 2.36fghi 0.55bcdefg 0.1h 126.41i 0. DW = dry weight.58abc 2.18j 0.9abcd 20.20gh 0.5abcde 126.97ab 0.40abcd 0. Growth parameter Shoot Treatment Plant height (cm) 116.82abcdefg 0.50abcdef 2.61d 0. and Psp = Pseudomonas Page 10 of 15 (page number not for citation purposes) .0ab 141.27abc 1.3–2802 μg/ml malic acid.39g 1.4fgh 135.biomedcentral. BIHB 811 was 22.31hij 2.84ef 6.39defgh 1.65cd 0.4fgh 130.24ij 0.24efg 1.81efg 0.96cde 0.52hi 0. respectively.4–114.34a 0.8a 146.57abc 7.79ab 6.5fg 20.72ab 2.17abcd 2.74bcd 0. 38.74bcd 0.1 and 29. 10.4cdefg Root DW (g/plant) 0.93bcdef 0.74bcd 0.84ef 4.58a 3.0–349.88abcd 2.8fgh 142.77bcd 0.51hi 1. and muriate of potash @ 80 kg K/ha to all the treatments.88abcd 5. poae BIHB 752 and Pseudomonas sp.61ef 4.56bcdefg 1.88a 0.38ef 4.63gh 1.89cdef 5.22abcd 0. Citric acid production shown by the two strains P.67a 3.27abcd 0.0efg 145.9–17.70cd 0.1 μg/ml succinic acid.47abcd 0.0abcde 126.03a 3.08i 0.48hi N P NP0K NPTCPK NPSSPK NPTCPK+Pt BIHB 728 NPTCPK+Pt BIHB 736 NPTCPK+Pt BIHB 745 NPTCPK+Pt BIHB 747 NPTCPK+Pt BIHB 749 NPTCPK+Pt BIHB 750 NPTCPK+Pt BIHB 757 NPTCPK+Pt BIHB 759 NPTCPK+Pt BIHB 763 NPTCPK+Pt BIHB 769 NPTCPK+Pp BIHB 730 NPTCPK+Pp BIHB 752 NPTCPK+Pp BIHB 808 NPTCPK+Pf BIHB 740 NPTCPK+Psp BIHB 751 NPTCPK+Psp BIHB 756 NPTCPK+Psp BIHB 804 NPTCPK+Psp BIHB 811 NPTCPK+Psp BIHB 813 1.92cde 0. Pp = P.53abcd 2.4bcdef 24.93ab 0.49bcdefg 2. Table 6: Influence of fluorescent Pseudomonas on growth and nutrient content of maize after 90 days in Environment Control Chamber.30hij 0.04bcde 4.79abcd 0. Pf = P.95ab 0.74bcd 0.84abcde 1.5bcdef 131.14def 4.57abc 2.58abc 2.32bcde 0. BIHB 756 was 26.84def 0.8 μg/ml lactic acid.5gh 141.0abcdefg 119.43ab 1.43efg 1.47ab 1. poae.93ab 0.51ef 7. Citric acid production observed for only Pseudomonas sp.63ab 0.77cdef 3.40a 0.1a 26.08bc 0.83abcd 4.31hij 0.55hi 0.51abcde 2.37g 1.72cdef 3.3 μg/ml lactic acid.63cdef 2.82abcd 0.55a Nutrient content (%) Root K N P K Root length (cm) 17. N and K applied as ammonium sulfate @ 240 kg N/ha.6ab 24.4cdefg 127.61ab 5.83abcd 0.37f 2.20gh 0.10abcde 1.25defg 0.91abc 3. 2549–6035 μg/ml gluconic acid.75ef 5.1abcde 131.26defg 0.7 μg/ml succinic acid.63cdef 3.60def 2.9defg 27.5bcdef 26.3fgh 130.43efg 1.85abc 0.39defgh 2.1 μg/ml oxalic acid.75cdef 2.6abcd 148.43cdefgh 2.58hi 0.BMC Microbiology 2009.22gh Values are the mean of 8 replicates.67cdef 1.16b 1.03f 4.31cde 0. fluorescens.5g 18.7 μg/ml 2-ketogluconic acid.95ab 2.10abcde 1.92bcdef 2.63abcdefg 0.23fg 0.5ab 26. trivialis. SSP = single super phosphate (120 kg P/ha).5 μg/ml.8–54.90abc 1.4 μg/ml formic acid. Organic acids during NCRP solubilization ranged from 8.39ab 0.76bcd 2. and 32.5abcd 25.9defg Shoot DW (g/plant) 4.47hi 0.9ab 142.32bcd 2.39defgh 0.59ab 2.27abc 1.49abcd 0.78abcd 0.64gh 0.8abc 24. 36.15bcd 2.8–50.98cd 1.95cd 0.01bcd 0.com/1471-2180/9/174 μg/ml 2-ketogluconic acid.63def 5.70abcdefg 0.60def 2.24defg 0.64abc 2.9defg 23.1–75.5fg 21.13abcde 2.62d 0.1–32.3 μg/ml during MRP solubilization.0abcde 141.23fg 0.35abc 0.11bc 1.40fg 1.66fgh 0.88de 0.20abcd 0.64a 0.03def 4.26defg 1.0abcd 26.82abcd 0.27cdef 0.68cdcd 0. respectively.86abc 0.6defg 143. Pseudomonas fluorescens BIHB 740 produced 59.74bcd 0.5ab 22.21gh 0.34ghi 2.3efg 20.25abcd 0.8abcd 22.01.70abc 8. 70. and 33–415 μg/ml malic acid.18abcd 0.4abcd 18. 9:174 http://www.63abc 0.62gh 0. 136.49cdefg 1.79abcd 0.0ab 23.25defg 0.83bcdef 2.56abc 3. Pt = P.50ef 2.83d 1.14hi 0.50cdefg 1. TCP = tricalcium phosphate (120 kg P/ha).95cd 1.82ef 7.30cdef 0.37efghi 2.10bcd 3.1cdefg 130.8abcd 24.47defg 1.60abc 2. 45.45abcd 0.55hi 1. Values with common letters in each column do not differ statistically according to Duncan's Multiple Range Test at p ≤ 0.46def 6.46ef 8.5abc 137.1abcd 24.3 μg/ml formic acid during NCRP solubilization.

The root length was significantly higher in fifteen PSB treatments over NP0K and thirteen PSB treatments over NPTCPK and NPSSPK. NP0K. The P content was significantly higher in NPSSPK over NP0K and NPTCPK. BIHB 751 and Pseudomonas sp. BIHB 740 and P. NPTCPK+Psp BIHB 756 and NPTCPK+Pf BIHB 740. The treatments NPTCPK+Pt BIHB 745. The final pH with non-significant difference among various treatments was less than the initial pH. NPTCPK and NPSSPK. 2a). and Pseudomonas sp. The root K was significantly higher in NPTCPK+Pt BIHB 745 and NPTCPK+Pt BIHB 728 over NP0K. Plant NPK content The treatments showed significant difference in the nutrient content of roots and shoots (Table 6). followed by NPTCPK+Pt BIHB 763. A non-significant difference in the shoot N was observed with NP0K. BIHB 811 showed dissimilarity in not producing malic acid. Soil properties The soil pH. 2c). The root dry weight was significantly higher in NPTCPK+Pt BIHB 749 over other PSB treatments. fluorescens BIHB 740 were formed at 2000 linkage distance. During MRP solubilization a single cluster including six sub-clusters and two single branches of P. Pseudomonas sp. NPTCPK and NPSSPK. P. The root N was significantly higher in eight PSB treatments over NP0K. trivialis BIHB 769 and P. Influence on plant growth Significant difference was observed for the growth parameters in maize among PSB treatments and uninoculated control treatments (Table 6). The highest shoot dry weight with NPTCPK+Psp BIHB 813 was statistically at par with NPTCPK+Pp BIHB 730. NPTCPK+Pt BIHB 745. The plant height was significantly higher in fifteen PSB treatments and NPSSPK over NP0K. 2b). BIHB 811 stood outside the cluster (Fig. NPTCPK+Pt BIHB 769. trivialis BIHB 763 stood independently outside the cluster (Fig. NPTCPK and NPSSPK. 9:174 http://www. NPTCPK+Pp BIHB 730 and NPTCPK+Psp BIHB 756. Pseudomonas sp. The maximum increase was obtained with NPTCPK+Pt BIHB 745. Nine PSB treatments exhibited significantly higher plant height over NPTCPK. K contents were significantly affected by PSB treatments (Table 7). The shoot K was significantly higher in NPTCPK+Psp BIHB 756. Other treatments were statistically at par with NPTCPK and NPSSPK. URP. Pseudomonas sp. In NCRP solubilization P. BIHB 751 differed from the other strains in producing oxalic acid. lactic. BIHB 811 in producing the lowest quantity of gluconic acid and the highest quantity of 2-ketogluconic. BIHB 751 and Pseudomonas sp. and NPTCPK+Pt BIHB 747 treatments. 2b). NPTCPK and NPSSPK. For TCP solubilization a single cluster was obtained at 2000 linkage distance. NPTCPK and NPSSPK. The highest N was obtained with NPTCPK+Pt BIHB 736. NPTCPK and NPSSPK. NPTCPK+Pt BIHB 769. 2). trivialis BIHB 747. while Pseudomonas sp. NPTCPK and NPSSPK. organic matter and available N. The shoot P was significantly higher in ten PSB treatments over NP0K. NPTCPK and NPSSPK. Plant height increase was the highest with NPTCPK+Pt BIHB 759. The treatments NPTCPK and NPSSPK were statistically at par with NP0K. BIHB 751 stood separately outside the cluster in producing the lowest quantity of gluconic acid and the highest quantity of malic acid (Fig. The shoot N was statistically higher in seven PSB treatments over NP0K and two PSB treatments over NP0K. BIHB 751 in the highest malic acid production. Pseudomonas sp. lack of succinic acid production. BIHB 811. followed by NPTCPK+Pt BIHB 745. The treatments NPTCPK and NPSSPK were statistically at par with NP0K. The highest P content obtained with NPTCPK+Pt BIHB 745. and producing the lowest quantity of gluconic acid and the highest quantity of 2-ketogluconic acid. NPTCPK+Pt BIHB 747 and NPTCPK+Pt BIHB 757 were statistically at par and showed significantly higher root dry weight over NP0K. BIHB 751 and P. poae BIHB 752 were observed at 2000 linkage distance. The shoot dry weight was significantly higher in seven PSB treatments over NP0K. while Pseudomonas sp. BIHB 751 differed in producing the lowest quantity of gluconic acid and the highest quantities of 2-ketogluconic and malic acids. NPTCPK+Pp BIHB 808. NPTCPK and NPSSPK. Pseudomonas sp.com/1471-2180/9/174 Cluster analysis based on the organic acid profiles during TCP. The highest decrease recorded with NPTCPK+Pt BIHB 757 was statisti- Page 11 of 15 (page number not for citation purposes) . Pseudomonas sp. NPTCPK+Pt BIHB 759 and NPTCPK+Pt BIHB 745 over NP0K. Pseudomonas trivialis BIHB 763 was separate from other strains in producing the highest quantities of gluconic and formic acids (Fig. and succinic acids. Increment in height was significantly higher with NPTCPK+Pt BIHB 759 and significantly lower with NPTCPK+Psp BIHB 751 over NPSSPK. The cluster incorporated 5 sub-clusters and separate branches of Pseudomonas sp. trivialis BIHB 759. In URP solubilization a single cluster of three sub-clusters and single branches of Pseudomonas sp. NPTCPK and NPSSPK. NPTCPK+Pt BIHB 757.BMC Microbiology 2009. The maximum increase was obtained with NPTCPK+Pt BIHB 736. The root P was significantly higher in three PSB treatments over NPSSPK. NPTCPK+Pt BIHB 747. Other PSB treatments were statistically at par with NPSSPK. P. NPTCPK+Pt BIHB 745.biomedcentral. MRP and NCRP solubilization generated Pseudomonas groups with strains belonging to the same or different species (Fig. trivialis BIHB 745 and P. The N content was statistically at par in NP0K. followed by NPTCPK+Pp BIHB 752 and NPTCPK+Psp BIHB 756. BIHB 811 stood outside the cluster as independent branches at 600 linkage distance (Fig 2d). Pseudomonas trivialis BIHB 747 differed in the highest gluconic acid production.

BIHB 751 than other Pseudomonas strains substantiated the involvement of gluconic acid in solubilization of calciumbound phosphates.049fgh P 0. NPTCPK and NPSSPK.027cde 0.85bcdef 3.6.25bcd 6. The production of 2-ketogluconic. Pseudomonas fluorescens and Pseudomonas spp.032i 0.36hi N 0.022efgh 0.43fghi 4.0017ijkl 0.13bcd 6.0016jkl 0. NPTCPK and NPSSPK.053fg 0.024def 0.042ijk 0. The highest available K was recorded for NPTCPK+Psp BIHB 756. MRP and NCRP could possibly be due to the higher gluconic acid production in presence of TCP.21bcd 6.0019ijkl 0.06abc 3.01.062de 0. B.93d 6. 5). The highest available P content was obtained with NPTCPK+Pt BIHB745 among PSB treatments and with NPSSPK among uninoculated treatments.057bcd 0.072b 0. The soil P content was significantly higher in three PSB treatments over NP0K. Enterobacter intermedium. Rhodococcus erythropolis and Serratia marcescens [3.028bcd 0. Succinic acid also appeared contribut- Discussion The organic acid production during solubilization of inorganic phosphates by the efficient phosphate-solubilizing strains of Pseudomonas trivialis.059e 0.0025defg 0. Pp = P.081a 0.17bcd OM (%) 3.074b 0. The available Ca was significantly higher in three PSB treatments than other PSB treatments.0024efgh 0.0021ghij 0.046fghi 0.044hij 0. N and K applied as ammonium sulfate @ 240 kg N/ha.057fg 0.070bc 0. cally at par with all other PSB treatments but significantly lower than NP0K. Citrobacter sp.0020hijkl 0.039jkl 0..049efgh 0. poae.069bc 0.022efg 0.017h 0.033ab 0. NPTCPK and NPSSPK. megaterium.0021ghij 0.82bcdefg 3.50abc 6.20.059bc 0. NP0K.0031bc 0. NPTCPK and NPSSPK.02cd 6.058ef 0.0019ijkl 0. succinic.0015kl 0.93d 6. None of Pseudomonas strains produced propionic acid unlike Bacillus megaterium strains during phosphate solubilization [3]. [16].037kl 0.00abcd 3. Delftia sp.055cde 0. Gluconic acid was the major organic acid produced as reported during phosphate solubilization by Pseudomonas sp.0030bcd K 0.027cde 0.19bcd 6. The lower production of gluconic acid and lower TCP solubilization by Pseudomonas sp.3.33abcd 6.026cde 0.080a 0. lactic.040jk 0.0022fghi 0.022efg 0.23bcd 6.74cdefgh 4. and Pseudomonas corrugata [6]..035a 0. Bacillus coagulans. and muriate of potash @ 80 kg K/ha to all the treatments. 9:174 http://www. [19]. P.065cd Values are the mean of 8 replicates. Azospirillum spp.55efghi 3. oxalic.061de 0. corroborated their involvement in phosphate solubilization [1. The organic matter content was significantly higher in four PSB treatments than NP0K.23bcd 5. Chryseobacterium sp..BMC Microbiology 2009.065a 0.033hi 0.17bcd 6. Citrobacter koseri. Pt = P.0024efgh 0.022efg 0.018gh 0.30abcd 6.030abc 0. fluorescens [17]. malic. Available nutrients (%) Treatment NP0K NPTCPK NPSSPK NPTCPK+Pt BIHB 728 NPTCPK+Pt BIHB 736 NPTCPK+Pt BIHB 745 NPTCPK+Pt BIHB 747 NPTCPK+Pt BIHB 749 NPTCPK+Pt BIHB 750 NPTCPK+Pt BIHB 757 NPTCPK+Pt BIHB 759 NPTCPK+Pt BIHB 763 NPTCPK+Pt BIHB 769 NPTCPK+Pp BIHB 730 NPTCPK+Pp BIHB 752 NPTCPK+Pp BIHB 808 NPTCPK+Pf BIHB 740 NPTCPK+Psp BIHB 751 NPTCPK+Psp BIHB 756 NPTCPK+Psp BIHB 804 NPTCPK+Psp BIHB 811 NPTCPK+Psp BIHB 813 pH 6.40ghi 3.062ab 0. SSP = single super phosphate (120 kg P/ha). TCP = tricalcium phosphate (120 kg P/ha). Pseudomonas poae.42fghi 4.049fgh 0. trivialis.025cde 0.072b 0.48efghi 3. The higher solubilization of TCP than URP.0026cdef 0.0038a 0. fluorescens.025cde 0. [18].060abc 0.13abc 3. NPTCPK and NPSSPK.027cde 0.025cde Ca 0.020fgh 0.052g 0.21].26abcd 6.24bcd 5.18bcd 6.6]. respectively.049efgh 0.18bcd 6.32a 3.029bc 0.0027cdef 0.0020hijk 0. formic and citric acid in small quantities by Pseudomonas strains have also been reported during phosphate solubilization by Arthrobacter ureafaciens.0015l 0.051efg 0. and Psp = Pseudomonas sp. NPTCPK and NPSSPK.0028cde 0. Pseudomonas fluorescens.63defghi 3.057fg 0.072b 0.63ab 6.069bc 0.039kl 0. The results indicated that the quantity of organic acids produced differed with the nature of phosphate substrates and Pseudomonas strains (Tables 2.046ghi 0.57efghi 3. Values with common letters in each column do not differ statistically according to Duncan's Multiple Range Test at p ≤ 0.17ab 4. NPTCPK and NPSSPK and statistically at par among NP0K.89bcde 3.019fgh 0.20bcd 6. The soil N content was significantly higher in five PSB treatments than NP0K.12cd 6. The soil K content was significantly higher in nine PSB treatments than other PSB treatments.90bcde 3.085a 0.29i 3.0033b 0. Pf = P.034l 0.038h 0.55efghi 3. Arthrobacter sp.050efg 0.biomedcentral.com/1471-2180/9/174 Table 7: Influence of fluorescent Pseudomonas on soil properties after 90 days in maize in Environment Control Chamber..052def 0.018gh 0.43fghi 3.16.79bcdefg 4. NP0K.0026defg 0.73a 6. Page 12 of 15 (page number not for citation purposes) . 3. 4.

The significantly higher plant growth and N. Bacillus circulans in mungbean [28] and Pseudomonas sp. UPR. and Serratia marcescens in maize [17].com/1471-2180/9/174 ing to TCP solubilization as it was produced by high TCPsolubilizing strains and not by low TCP-solubilizing Pseudomonas sp. Pseudomonas strains exhibiting high TCP solubilization in vitro differed significantly in enhancing the plant growth in the soil indicating interplay of some other growth factors besides phosphate-solubilization (Tables 2. phosphate-solubilizing microorganisms improve plant health directly by the production of phytohormones [31]. 4. 4. 6. physiological and growth conditions of the culture [26]. Pseudomonas fluorescens in peanut [27]. The higher solubilization and lowered quantities of organic acids detected in the presence of NCRP could be due to the higher reactivity and greater diversion of organic acids in the neutralization of free carbonates in the solubilization of NCRP as compared to MRP and URP [23. gluconic.24]. 2). The increased and continuous P availability in the soil promotes biological nitrogen fixation [27]. Likewise. BIHB 751 strain. Previously also the quantities of solubilized phosphorus could not be correlated with the quantities of organic acids in the culture medium [22]. Phosphate solubilization is a complex phenomenon which depends on the nutritional. strain BIHB 751 producing the highest quantity of 2-ketogluconic acid but showing the lowest TCP and URP solubilization also differed from Enterobacter intermedium reported for the enhanced phosphate solubilization with increasing 2-ketogluconic acid production [21]. No correlation among TCP solubilization. The lack of oxalic acid production by efficient phosphate-solubilizing Pseudomonas strains signified non involvement of oxalic acid in TCP solubilization though this acid has been implicated besides citric. and 7). 5). Likewise. no relationship could be ascertained between the quantity of organic acids produced and the solubilization of rock phosphates by Pseudomonas strains as the highest solubilization observed for NCRP among the rock phosphates was coupled to the lowest production of total organic acids (Tables 3. 3. However. increased soil P availability and higher plant P content would be useful particularly in the cold deserts of Lahaul and Spiti where soil P deficiency is attributed mainly to the reaction of P with calcium carbonate and calcium sulphate forming insoluble di. The inorganic acids and H+ ions of microbial origin and H+ ions released from the plant roots during ammonium assimilation are also reported to influ- Page 13 of 15 (page number not for citation purposes) . MRP and NCRP have fluorapatite structure with the highest substitution of phosphate with carbonate in NCRP [23]. Plant-microbe interaction is a complex phenomenon with the interplay of several mechanisms and environmental factors.BMC Microbiology 2009. The bacterial strains also differ in utilizing root exudates in producing biologically active substances and root colonizing ability known to influence the plant growth-promoting action of rhizobacteria [35]. P and K contents in maize with PSB treatments underlined the advantage of phosphate-solubilizing activity of microorganisms for plant growth promotion (Table 6 and 7). The decrease in soil pH in PSB treatments indicated the production of organic acids by Pseudomonas strains as also reported for phosphate-solubilizing Aspergillus niger and A. BIHB 751 showing the lowest TCP solubilization suggested that phosphate solubilization is an important mechanism of plant growth promotion. P. The strains standing outside the clusters differed qualitatively and/or quantitatively from other strains in the production of organic acids (Tables 2. Apart from making P available to the plants. The increased growth and P uptake have been reported on PSB inoculations with Pseudomonas sp. Pseudomonas sp. in wheat [29]. lactic and succinic acids in phosphate solubilization in alkaline vertisols [20]. The results implied that Pseudomonas strains are independent of their genetic relatedness in their phosphate-solubilizing ability and organic acid production even under similar set of culture conditions. However. The enhanced growth and higher N.9]. production of organic acids and plant growth promotion could be established as the highest solubilization and plant growth promoting activity was observed for P. The strains clustered together and those standing outside the clusters or sub-clusters belonged to different Pseudomonas species characterized previously by 16S rRNA gene sequencing [8. Cluster analysis of organic acid profiles generated different groups revealing inter and intra-specific variation in the production of organic acids by Pseudomonas strains (Fig. The TCP solubilization in soil by fluorescent Pseudomonas strains as evidenced by in vitro TCP solubilization.and tricalcium phosphates.biomedcentral. tubingensis [36]. and K content in plant tissues and soil with some PSB treatments over NPSSPK might be due to the immobilization of applied P by native soil microbiota and physico-chemical reactions in the soil. the higher solubilization and higher production of organic acids in the presence of TCP could be attributed to its amorphous nature with simple structure and absence of any free carbonates as compared to the crystalline lattice structure of the rock phosphates [25]. Pseudomonas strains have been reported to vary in their ability for phytohormone production [32-34]. trivialis BIHB 745 not showing the highest organic acid production. the lowest organic acid production and plant growth promotion by Pseudomonas sp. 9:174 http://www. The rock phosphates recommended for acid soils are reportedly not effective in alkaline soils as P source for the crops [30]. less pH decline in soil during plant growth promotion experiments than phosphate solubilization in culture medium could be due to the buffering nature of soil [20]. 5).

58:425-434. U. 13. India. Vyas P. World Journal of Microbiology and Biotechnology 1998. Young CC: Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities. trivialis BIHB 745. Dev. 9. Vyas P. Sharma. Whiting PH. CSK HP KV (Agriculture University). Bolland M: Effectiveness of rock phosphates. Shen FT. 1978. 1954. 2007:215. Current Microbiology 2008. Biochemical Journal 1976.P. Hwangbo H. Chen YP. 56:168-174.BMC Microbiology 2009. Mehta RK: Identification of organic acids produced during rice straw decomposition and their role in rock phosphate solubilization. Significant difference in plant growth promotion among the efficient phosphate-solubilizing Pseudomonas strains point at the need for selecting the potential strains based on plant growth promotion in the soils supplemented with insoluble phosphates for their targeted application. Naresh Kumar G. Simard PR. Illmer P. Narayanasamy G. 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