Journal of Mıcrobıology and Mıcrobıal Research Vol.1(1),pp.1-6, May, 2013 http://www.peakjournals.org/sub-journals-JMMR.html ©2013 Peak Journals
Full Length Research Paper
Characterization of rock phosphate-solubilizing microorganisms isolated from wheat (Triticum aestivum L.) rhizosphere in Mali Amadou Hamadoun Babana*, Amadou Hamadoun Dicko, Kadia Maïga and Diakaridia Traoré
Accepted 25 May, 2013 In this study, we focused on the characterization of six strains of bacteria and two phosphate solubilizing fungal strains isolated from wheat rhizosphere in Mali. Two bacteria strains (Pseudomonas sp. BR2 and non-identified bacteria B3), which did not form halozone around colonies, were able to release phosphorus from Tilemsi rock phosphate in liquid medium. Based on solubilization index (SI), Vibrio splendidus B27 was the most efficient rock phosphate solubilizer on NBRIP agar plates (SI = 3.60). In liquid medium, the maximum phosphorus (P) solubilization was recorded by Pseudomonas sp. BR2 (60 mgl) for bacteria and Penicillium chrysogenum thom C13 (298.9 mgl) for fungi. Among nine different organic acids produced by the tested microorganisms, only citric acid showed a high significant positive correlation with P solubilized. In spite of the fact that no bacterium was able to produce cyanhydrique acid, Pseudomonas sp. BR2, Agrobacterium tumefacians BR10 and nonidentified bacteria B3 produced siderophores during their growth. Key words: Rock phosphate, phosphate-solubilizing microorganisms, wheat, characterization, Mali.
Laboratory of Research in Microbiology and Microbial Biotechnology (LaboREM-Biotech), Faculty of Sciences and Techniques, University of Sciences, Techniques and Technology of Bamako, BP E 3206, Mali. *Corresponding author. E-mail:
[email protected],
[email protected]. Tel: +22376124173. Abbreviations: TRP, Tilemsi rock phosphate; P, Phosphorus; PSM, phosphate-solubilizing microorganisms; PSB, P-solubilizing bacteria; PSF, Phosphate-solublizing fungi; IAA, indoleacetic acid; SI, Solublization index; PGPR, plant growth promoting rhizobacteria plants.
INTRODUCTION Phosphorus (P) deficiency is a major constraint to crop, particularly wheat production in Mali. However, Tilemsi rock phosphate (TRP), which deposits are estimated to be between 20 and 30 Million tons, is an important source of P for farmers (Bationo et al., 1997). Several research results clearly indicated that the direct application of TRP could be profitable in comparison with recommended imported P fertilizers (Bationo et al., 1997). However, the use of TRP as phosphorus source is limited by its inefficiency in many agricultural soils. While, many soil microorganisms, including bacteria and fungi, are able to mobilize sparingly soluble inorganic and organic phosphates, and they have an enormous potential in providing soil phosphates for plant growth (Babana, 2003; Hamdali et al., 2012).
With the aim of improving the response of crop cultivated in Mali to fertilization with TRP, we recently isolated bacteria and fungi with great potential to solubilize TRP (Babana and Antoun, 2006). In the present work, we describe the characterization of six TRP-solubilizing bacteria and two TRP-solubilizing fungi, isolated in the rhizosphere of wheat cultivated in Mali and selected for high capacity to release phosphorus from rock phosphate.
MATERIALS AND METHODS Phosphate-solubilizing microorganisms Six
strains
of
bacteria
(B22 = Agrobacterium
Babana et al.
tumefaciens, B27 = Vibrio splendidus, BR2 = Pseudomonas sp., BR10 = Agrobacterium tumefacians/radiobacter, B3 = non identifié and BR8 = non-identified) and two fungi (C1 = Aspergillus awamori nakazawa and C13 = Penicillium chrysogenum thom) isolated from the rhizosphere of three irrigated wheat (Babana and Antoun, 2006) were used in this study.
Solubilization index (SI) To determine the solubilization index of the phosphatesolubilizing microorganisms (PSM) used in this work, 0.1 ml of each PSM culture preserved in sterile distilled water was placed on NBRIP agar (Nautiyal, 1999) plates [containing insoluble glucose, 10 g; MgCl2.6H2O, 5 g; MgSO4.7H2O, 0.25 g; KCl, 0.2 g and (NH4)2 SO4, 0.1 g; TRP, 5 g; agar, 15 g and the pH was adjusted to 7.0 and dissolved in 1000 mL distilled water] and incubated for seven days. Solubilization index was calculated as (colony diameter + halozone diameter) colony diameter (Edi-Premono et al., 1996).
Bacterial growth, organic acid production and rock phosphate solublization Bacterial growth, organics acids production and phosphate solublization were determined using TRP (5 g/L) in NBRIP broth medium. Three culture replicates 6 were inoculated with 10 ufc /ml of each P-solubilizing 6 bacteria (PSB) and 10 spores of PSF/ml and grown for seven days at 28°C on a rotary shaker (180 g/min) in 250 ml Erlenmeyer flasks containing 50 ml of liquid NBRIB broth medium. A sample of the growth medium was collected everyday for further analysis.
Bacterial growth Bacterial growth was assessed by determining the mycelium dry weight of fungi after drying at 70°C, and the total bacterial protein contents after total hydrolysis of bacterial cells with 0.1 N NaOH during 60 min at 100°C (Lowry et al.,1951).
Organic acids production For the analysis of organic acids, bacterial cultures were filtrated through 0.22 µm filter (Millipore, GTBP) and 20 ml of filtrates were injected to HPLC (Model: Hitachi L5000) equipped with a Hitachi L-3000 Poto Diode Array detector. The organic acid separation was carried out on Aminex HPX-87C column (Bio-Rad Laboratories, Inc.) with 10.8% acetonitrile in 0.0035 M H2SO4 as mobile
2
phase. Retention time of each signal was recorded at a wavelength of 210 nm. The pH of the supernatant was determined using a pH-meter.
Rock phosphate solublization For soluble phosphorus content analysis, the cultures were sampled each day, centrifuged at 10,000 × g for 10 min and the pH of the supernatant measured. Phosphorous concentrations were measured using a spectrophotometer (Thermo scientific, Genesis 20) at 880 nm by the vanadate-molybdate method (Tandon et al., 1968). Similar measures were carried out in noninoculated flasks incubated under the same conditions.
Production of growth substances Siderophore excretion Siderophore production by the tested microorganisms was determined on the Chrome Azural S (CAS) medium (Schwyn and Neilands, 1997), modified by Meaningn and Mattila-Sandholm, 1994. Microbial growth on this medium was verified every day and expressed as the number of day necessary for a tested microorganism to cover all the petri dish. The reaction of the blue-CAS was determined by measuring the distance on which color changes occurred from the inoculation site. The size of the zones and the intensity of the color change were estimated and compared to the controls.
Production of cyanhydric acid The capacity of the tested PSB and poly-silicic-ferric (PSF) to produce chyanidric acid was verified using the method described by Bakker and Schippers (1987). Every tested PSB or PSF was used to inoculate a petri dish containing medium composed with: tryptic soy broth (TSB, Difco), 3 g; agar, 20 g; glycin 4.4 g and distilled water, 1 L. A filter paper (9 cm diameter) containing picric acid, 0.5% and Calcium carbonate 20%, was placed on the lid of each petri dish in inverse position. Petri dishes were verified each day to identify the ones containing filter papers having changed color from orange to brown, indicating cyanhidric acid (HCN) production.
Production of indole acetic acid (IAA) The solid Luria-Bertani medium enriched with tryptophan (Bric et al., 1991) was used to identify indoleacetic acid (IAA) producing microorganisms. For that, all the isolates were inoculated on a nitrocellulose membrane. Bacteria
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Table 1: Rock phosphate solublization by phosphate-solubilising microorganisms as expressed by Halozone and solubilization index.
Microorganisms Halzone + colony (mm) BR2 3.1 BR8 5.94 BR10 5.99 B3 3.8 B22 3.82 B27 6.92 C1 42.15 C13 22.23
Colony (mm) 3.1 2.94 1.99 3.8 1.82 1.92 25.12 12.23
Halozone (mm) 0 3 4 0 2 5 17 10
Solubilization index (SI) 1 2.02 3.01 1 2.1 3.6 1.68 2.16
Values are means of triplicates.
Table 2. P solubilized by the PSM isolates, the pH and the titrable acidity of the culture media.
Sources of variations Repetitions PSM isolates Error
df 2 7 14
Means squares P solubilized pH 0.0000ns 0.0007ns 3.19*** 2.7*** 0.004 0.009
Titrable acidity 0.0001ns 1.5*** 0.004
***Significant at p < 0.001, ns = statistically not significant.
which synthesize IAA are identified by the formation of a characteristic red halo which encircles the colony. RESULTS Solublization index (SI) Based on colony diameter and holozone, SI was calculated for each isolate and results are presented in Table 1. Results showed that B27 was the most efficient rock phosphate solubilizer on NBRIP agar plates (SI = 3.60) among rock phosphate-solubilizing bacteria. Whereas C13 showed highest solubilizing index (SI = 2.16) among fungi. On containing agar plates, TRPsolubilizing microorganisms formed clear zones by solubilizing suspended rock phosphate. Halozone formed by rock phosphate-solubilizing microorganisms ranged from 0-5 mm for bacteria and 1217 mm for fungi (Table 1). BR2 and B3 did not produce halozone while growing on solid medium with TRP as sole phosphorus source. The SI of the most efficient bacteria was greater than that of the fungi although fungi produce large halozone compared to bacteria. Bacterial growth, organic acids production and rock phosphate solublization The analysis of variance (Table 2) showed significant
differences between the tested PSM isolates regarding their capacity to solubilize inorganic phosphate, modify the pH and increase the titrable acidity of the culture media. The pH, organic acids, amounts of soluble-P and bacterial growth after seven days of incubation are presented in Table 3. In the blank treatment, no soluble-P was detected and no pH decrease was observed. The maximum amount of solubilized P and the time required to solubilize P varied with isolates and were between 18.17 and 298.9 mg/l. All isolates reached their maximum efficiency between 5 to 7 days of incubation. For bacteria maximum, P solubilization was recorded by Pseudomonas sp. BR2 (90 mg/l) while, for fungi maximum P solubilization was recorded by P. chrysogenum thom C13 (298.9 mg/l), followed by A. awamori nakazawa (181.27 mg/l) with a maximum drop in the pH to 3.64. The minimum concentration of soluble-P (18.17 mg/l) was observed in the cultures of V. splendidus B27 and the pH of the medium was relatively high (5.62). Even though maximum drop in pH was associated with higher production of organic acids, in some cases, for example, C13 maximum drop of pH was associated with lower production of organic acids. No halozone was detected around colonies of BR2 and B3 during the dissolution test performed on solid medium although these bacteria were isolated on this basis. However, despite the absence of halozone around BR2 and B3 on solid medium, both
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Table 3. Phosphorus solubilized, final pH, organic acids produced, titrable acidity and microbial growth following seven days of incubation.
Isolates
P solubilized (mg/ml)
α
β
pH
Organic acids (g/l)
Titrable acidity (meq/50 ml)
Microbial growth
Proteins concentration (µg/ml) 88 ± 2 60 ± 1.7 66 ± 3 53 ± 2 62 ± 2.7 47 ± 2.7
Bacteria BR2 BR8 BR10 B3 B22 B27
90.01c 27.11e 33.11d 20.08f 29.96d 18.17f
4.2b 4.97e 4.67d 5.24f 4.59c 5.62g
5.56b 2.96d 5.63b 0.00g 3.70c 0.58e
2.08d 2.19c 2.08d 1.32f 1.76e 1.40f
Mycelia dry weight (mg/50ml) 272 ± 2,6 209 ± 1
Fungi C1 C13 F test
298.86a 181.27b ***
4.92e 3.64a ***
16.88a 0.24f ***
3.53a 2.58b ***
α P solubilized 7 days after inoculation (values are means of 3 replications); β pH of the culture media after 7 days of growth. *** Significant if at 0.00. In the same column, means with the same letter are not significantly different at p0.05.
Table 4. Organic acids produced in culture medium by PSM after twelve days of incubation.
PSM*
Organic acids (g/l) Pyruvic Succinic Tartric 1.19 5.57 10.77 7.11 0.17 0.24
Gluconic 13.28 3.16 -
Acetic 3.09 2.71 -
Formic 0 -
C1
9.08
11.47
8.56
6.42
21.73
C13 LSD** (0.05)
14.4 0.78
0.03
0.027
0.017
0.74
BR2 BR8 B3 B22 B27
Oxalic 7.73 3 3.54 4.7 0.08
Citric 3.83 2.33 12.44 1.71 1.42
aconitic 1.78 1.93 2.07 -
12.61
0.09
51.48
23.44
0.18 0.04
6.92 0.033
15.47 0.04
0.16 0.03
*Phosphate-solubilizing microorganisms. **Least Significant difference.
bacteria could solubilize rock phosphate in liquid medium. BR2 solubilized even more phosphorus than all the PSB in this work. Organic acids produced by the PSM isolated from Wheat (Triticum aestivum L.) rhizosphere, are presented in Table 4. Results showed the presence of organic acids in the culture mediums of all the microorganisms used in this work (Table 4). Nine different organic acids were produced by the eight microorganisms used in this work,
among the organic acids produced, 3 were produced by all the microorganisms (citric acid, oxalic acid and tartric acid). In this experiment, all the PSM tested produce one or a mixture of organic acids. A. amowari nakazawa
C1 (16.88 gL) was the highest acid producing strain, and the lowest was B27 (0.19 gL). Most common acids produced were citric, oxalic and tartric acid; while formic acid (8.56 gL) was produced only by A. amowari nakazawa C1. This fungus produced highest concentration of citric acid (55.1 gL) and aconic acid (23.44 gL) and was the second oxalic acid (5.92 gL) producer in this work. Oxalic acid production by Enterobacter agglomerans has been reported by Kim et al. (1998). In this work, gluconic acid was produced at a concentration of 14.4 and 13.28 gL, respectively by P. chrysogenum thom C13 and Pseudomonas sp. BR2. Besides citric acid, oxalic acid and gluconic acid; fumaric acid was produced by all the tested PSM. Except BR8 and
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B27, aconic acid was produced by all the TRPsolubilizing microorganisms tested.
Correlation coefficients Correlation coefficient values showed least negative correlation (r = -0.25) between P solubilized and solublization index. This result means that a PSM who did not show halozone in solid medium can significantly release phosphorus from rock phosphate in liquid medium. This finding is not in agreement with what was reported by Illmer and Schinner (1992), but confirmed the conclusion from Johri et al. (1999) who supports that criterion for isolation of efficient PSM based on formation of a visible zone on agar plates was not a reliable technique. Phosphorus (P) solubilized was significantly correlated with colony diameter (r = 0.97) and the total diameter of colony and halozone (r = 0.96). Significant (p 0.05) positive correlation (r = 0.89) was found between P solubilized and halozone. Least negative correlation was obtained between solubilization index and organic acids produced (r = -0.19). This is due to the fact that perhaps organic acids diffuse slightly on solid medium or are, in great number, not efficient in rock phosphate solublization. A significant positive correlation was observed between P solubilized and organic acids produced by PSM (r = 0.90) suggesting that organic acids may play an important role but are not the only possible mechanism for P solublization. Drop in pH; for example microbial respiration, was observed by Illmer and Schinner (1992). Significant correlations were found between bacterial growth and organic acids (r = 0.92) organic acids and titrable acidity (r = 0.88) and titrable acidify and P solubilized (r = 0.93). A significant positive correlation was observed between citric acid production and phosphate solublization (r = 0.75).
Production of siderophore, indole acetic acid and cyanhydric acid Among microorganisms used in this work, only bacteria BR2 and BR10 produce siderophores in large quantities thus confirming the results of Chabot et al. (1993) who detected the production of siderophores by phosphatesolubilizing microorganisms isolated from different soils of Quebec. These bacteria have been known as plant growth promoting rhizobacteria plants (PGPR). Bacteria B3, also produces siderophores but in lower quantity than BR2 and BR10. Siderophores are substances that promote plant growth by inhibiting plant pathogens or deleterious. In addition to siderophore production, bacteria BR2, B22, B3 and BR10 produce IAA. During these tests, no bacteria could produce cyanhydric acid.
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DISCUSSION No halozone was detected around BR2 and B3 colonies during the rock phosphate-solubilization tests performed on solid medium, although these bacteria were isolated on this basis. This can, perhaps, be explained by the low diffusion of the organic acids produced by these bacteria during their growth or the use of a solubilization process without organics acids. However, despite the absence of halozone around these bacteria, both could solubilize rock phosphate in liquid medium. These results support those of Nautiyal (1999) and Johri et al. (1999), indicating that the isolation criterion of PSM based only on the formation of a halozone on solid medium is not an infallible criterion. This suggests that it would be preferable. Before selecting a PSM to be used as inoculant, we must determine, in addition to the halozone, the amount of P solubilized in liquid medium. Out of the eight PSM tested, three were produce by all the microorganisms (citric acid, oxalic acid and tartric acid). This finding conform to that of Nautiyal et al. (1999), who stated that more organic acids are produced by phosphate solubilizing microorganisms when glucose is used as carbon source. However, the PSM which produce more citric acid dissolved more phosphorus from rock phosphate suggesting that citric acid is most effective in rock phosphate-solubilization. This result is in agreement with those of Kang and al. (2008) and Reyes et al. (1999, 2001) indicating that the solubilization of different rock phosphates by Aspergillus sp. PS 104 and ++ Penicillium rugulosum Mps is mainly due to the production of organic acids. They also indicated that citric acid seemed to be the main acid in question. Instead, Fenice et al. (2000) observed that the mass production of gluconic acid is accompanied with inorganic phosphate solubilization by Penicillium variable P16 encapsulated. A significant positive correlation was observed between citric acid production and phosphate solublization (r = 0.75). This result is similar to the result obtained by Cunningham and Kuiack (1992) who reported that citric acid was a major organic acid produced by Penicillium bilaji in phosphate solubilization. In this work, we found that gluconic acid was produced by P. chrysogenum thom C13 and Pseudomonas sp. BR2. This finding is different from results Whitelaw et al. (1999) indicating that gluconic acid was not produced by PSM strains used in their experiments. However, it agrees with the reports by Illmer and Schinner (1992) indicating small quantities of gluconic acid produced from Aspergillus niger. None of the eight PSM in the present work produced lactic acid. However, Venkateswarlu et al. (1994) detected lactic acid from A. niger. In this work, we found that the PSM tested produced more citric acid in the presence of ammonium as nitrogen source. This result is similar to that of Cunningham and Kuiack (1992) who
Babana et al.
reported that citric acid was a major organic acid produced by Penicillium bilaji when nitrogen source was nitrate but it was not detected when ammonium was used. Among microorganisms used in this work, only BR2 and BR10 produced siderophores in large quantities, thus confirming the results of Chabot et al. (1993) who detected the production of siderophores by phosphatesolubilizing microorganisms isolated from different soils of Quebec.
Conclusion Rock phosphate-solubilizing microorganisms showed variation in their biochemical characteristics. Organic acid production was perhaps not the only possible reason for phosphorus solublization. Present study showed that Pseudomonas sp. BR2 is the best rock phosphatesolubilizing bacteria and A. amowari nakazawa and P. chrysogenum thom are the most efficient rock phosphatesolubilizing fungi on the basis of their P solubilizing activity. This study also showed that citric acid is highly implied in rock phosphate-solubilization while Pseudomonas sp. BR2, which solubilized rock phosphate and produced siderophores, indole acetic acid and cyanhydric acid, seems to be a PGPR
ACKNOWLEDGEMENTS We thank the Programme Canadien de bourse de la Francophonie for the fellowship, the Academy of Sciences for the Developing World (TWAS) and the “Agence Universitaire de la Francophonie (AUF)” for their financial contributions. REFERENCES Babana AH (2003). Mise au point d’un inoculant biologique pour le blé irrigué du Mali. Thèse de doctorat. Université Laval, Québec, Canada. Babana AH, Antoun H (2006). Effect of Tilemsi phosphate rock solubilizing microorganisms on phosphorus-uptake and yield of field grown wheat in Mali. Plant and Soil 287:51-58. Bakker AW, Schippers B (1987). Microbial cyanide production in the rhizosphere in relation to potato yield reduction and Pseudomonas spp-mediated plant growth-stimulation. Soil Biol. Biochem. 19(4): 451-457. Bationo A, Ayuk E, Ballo D, Koné M (1997). Agronomic and economic evaluation of Tilemsi phosphate rock in different agro ecological zones of Mali. Nutrient Cycling Agrosyst. 48:179-189.
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