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Effet du stress salin sur la croissance et la solubilisation du phosphate par des rhizobactéries
Elmeddah, Nawal; Kassous, Amina
Date:
2016
Résumé:
The use of microorganisms from rhizosphere is the best way to improve biological
solubilization of phosphorus in saline soil to reduce salt stress which can affect growth and
activity of rhizobacteria. The aim of our present study is to evaluate the effect of salt stress on
the growth and phosphate solubilization of phosphate tricalcique by five phosphate
solubilizing bacteria isolated from saline soil (R1, R2, R3, R4, R5) usingincreasing
concentrations of NaCl(0, 100, 200…...900mM). The ability of phosphate solubilization by
the five isolates has been tested on PVK solid medium where the R3isolate was considered to
be the most effective one with a maximum efficiency of 366.66%. The salt tolerance of
bacterial isolates in nutrient broth revealed variable results of optical density, the value of the
highest optical density corresponding to maximum growth was obtained from the isolate R4
(DO: 1,604) with 400 mM of NaCl. The quantities of released phosphorus by bacterial
cultures in NBRIP liquid medium under salt stress ranged from 135, 429μg /ml and
879,714μg. The highest rate was detected with isolates R2 and R4 with a significant amount of
soluble phosphorus of 879,714μg/ml and 834,476μg/ml respectively with 300 and 400 mM
of NaCl. pH values of bacterial cultures ranged from 5.337 to 3.72 were obtained which with
increasing of phosphate solubilization rate. The results obtained show that the bacterial
isolates used in this study tolerated high levels of salt and were able to solubilize insoluble
phosphate even at high NaCl concentrations.
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Méchali, Roufaїda; Kherrat, Soumia (2016)Phosphate solubilizing bacteria play a vital role in soil fertility, which they are used to promote the growth of a large group of plants.The aim of this work is the evaluation the capacity of bacterial isolates to release ...
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CHIBANI, HIBA RAHMAN (Malaysian Journal of Microbiology, Vol 13(2) June 2017, pp. 124-131 Malaysian Journal of Microbiology Published by Malaysian Society for Microbiology (In since 2011) 124 ISSN (print): 1823-8262, ISSN (online): 2231-7538 *Corresponding author Screening and characterization of plant growth promoting traits of phosphate solubilizing bacteria isolated from wheat rhizosphere of Algerian saline soil Chibani Hiba Rahman1*, Bouznad Ahcene1, Bellahcene Miloud2, Djibaoui Rachid1 1Laboratory of Microbiology and Plant Biology, Faculty of Natural and Life Sciences, University of Mostaganem, Algeria. 2University Center of Ain Temouchent, Algeria. Email: hiba.chibani@univ-mosta.dz Received 16 August 2016; Received in revised form 10 September 2016; Accepted 7 November 2016 ABSTRACT Aims: The capacity of some soil microorganisms to solubilize in soil is an important activity exhibited by plant growth promoting rhizobacteria (PGPR) to increase plant performance. This study aimed at isolation and selection of phosphate solubilizing bacteria from saline soil and in vitro evaluation of their plant growth promoting traits. Methodology and results: Phosphate solubilizing bacteria isolated from wheat rhizosphere, of saline soil in western region of Algeria were tested for their plant growth promoting traits such us indole acetic acid (IAA), hydrogen cyanide (HCN), siderophore and ammonia production and their ability to fix nitrogen. Among 104 bacterial isolates, 41 were selected for their phosphate solubilizing activity using tricalcium phosphate (TCP) as a sole phosphorus source. IAA production was shown by almost all the bacterial isolates. Twelve isolates were recorded positive for HCN production, 32 produced siderophore and 31 were able to fix nitrogen. The most dominant phosphate solubilizing bacteria found were identified as Pseudomonas followed by Aeromomas hydrophila Bacillus sp. and Burkholderia cepacia. Conclusion: Phosphate solubilizing bacteria that were isolated from saline soil showed a high potential in to producing growth promoting traits and can be used as inoculants to increase the phosphorus uptake by plants. Key words: Phosphate solubilising bacteria, saline soil, tricalcium phosphate, plant growth promoting traits INTRODUCTION Phosphorus is considered to be the most important element in plant nutrition of, after nitrogen. It is an essential component in all main metabolic procedures in plants for example energy transfer, photosynthesis, signal transduction and respiration (Khan et al., 2010). Inorganic phosphorus is found in soils, mostly in insoluble mineral complexes such as tricalcium phosphate Ca3 (PO4)2, ion phosphate FePO4 and aluminium phosphate AlPO4 (Barber, 1995), which appear after repeated applications of chemical fertilizers. Plants have not the capacity to absorb these insoluble forms besides only 0.1% of total phosphorus is in soluble form and it is available for plant nutrition (Zhou et al., 1992). It is for this reason that the available phosphorus levels have to be supplemented in most agricultural soil by adding chemical phosphorus fertilizers. The frequent and imprudent applications of chemical phosphorus fertilizers lead to the decrease of soil fertility by perturbing microbial population thus reduce crops yield (Gyaneshwar and Naresh, 2002). Among chemical environmental stress soil salinity is the most important stress factor for plants (Idikut et al., 2012). Salinity leads to osmotic stress and causes the formation of reactive oxygen species (ROS) thus disturbs cellular structures which and consequently damage mitochondria and chloroplast (Mittler, 2002). Soil salinity considerably reduces absorption of mineral nutrients, particularly phosphorus for the reason that phosphate ions precipitate with calcium ions in saline soil and become inaccessible to plants (Grattan and Grieve, 1999). Utilization of phosphate solubilizing bacteria to solve this problem for raison of their ability to solubilize phosphate in soil is supported by many researchers (Khan et al., 2007). Mechanisms like organic acid production, chelation, and ion exchange reactions are implemented by these bacteria to solubilize inaccessible phosphorus forms and make them available to plants (Vessey, 2003). Phosphate solubilizing bacteria (PSB) are part of plant growth promoting rhizobacterias (PGPRs) and are capable of solubilizing inorganic phosphate from different compounds, such as dicalcium phosphate, tricalcium phosphate and rock phosphate. Moreover, PSMs may also showed plant growth promoting traits such as indole acetic acid (IAA), cytokinins, gibberellic acid and ethylene production, hydrogen cyanide (HCN) and siderophore Mal. J. Microbiol. Vol 13(2) June 2017, pp. 124-131 125 ISSN (print): 1823-8262, ISSN (online): 2231-7538 production, nitrogen fixation and resistance to soil pathogens etc (Cattelan et al., 1999). The main objectives of this study were the isolation and screening of phosphate solubilizing bacteria from wheat rhizosphere of salt affected soil and in vitro assessment of their plant growth promoting activities. MATERIALS AND METHODS Soil sampling Saline soil samples were collected randomly from the rhizosphere regions of wheat plants growing at different sites at Relizane (western Algeria). All samples were kept in plastic bags and transported to the laboratory and stored at 4 °C. Total of fifteen soil samples were air-dried, ground and passed through 2 mm sieve before chemical analyses pH, soil moisture and conductivity of the soil samples were measured. Isolation of total phosphate solubilizing bacteria Isolation of PBS bacteria was carried out by serial dilution method. Ten grams of rhizosphere soil was dissolved in 90 mL of saline phosphate buffer, then shook for 30 min. One mL of rhizosphere soil suspension was added to 9 mL of sterile saline water to obtain a suspension with a 10-2 dilution. Subsequently 0.1 mL of the suspension diluted to 10-5 was grown on Pikovskaya (Pikovskaya 1948) PVK agar supplemented by 5 g of tricalcium phosphate (TCP) as a sole phosphorus source. Inoculated plates were incubated for 7 days at 28± 2 °C. Appearance of clear halo zone on Pikovskaya’s agar plates indicates positive phosphate solubilization ability. Bacterial colonies surrounded by a transparent halo on PVK agar were picked off, checked for purity and classified as putative P-solubilizers. Quantitative Estimation of Phosphate Solubilization in Culture Broth The isolated bacteria presenting halo zones on solid PVK medium were used to measure phosphate solubilization in liquid medium. The in vitro phosphate solubilizing capacity of each strain was determined on National Botanical Research Institute’s Phosphate growth medium (NBRIP) (Nautiyal, 1999) supplemented by 5 g of TCP as sole phosphorus source. The flasks containing 50 mL of NBRIP medium was inoculated with 1 mL of bacterial suspension (2×109 cfu/mL) in triplicates and incubated at 28±2 °C on a rotary shaker 180 rpm for 7 days. The cultures were harvested by centrifugation at 6000 rpm for 30 min. The phosphorus in supernatant was estimated by vanado-molybdate-yellow colour method (Jackson, 1973). The total soluble phosphorus was calculated from the regression equation of standard curve. The values of soluble phosphate liberated were expressed as μg ml-1 over control. The pH of culture supernatants were also measured using a pH Meter. Production of indole acetic acid (IAA) Production of auxin indole-3-acetic acid (IAA) by bacteria was tested using Lauria Bertani (LB) and Salkowski reagent. Fifty milliliter of Lauria Bertani (LB) containing (0.1g/L of L-tryptophan was inoculated with 1 mL of bacterial suspension (approximately 109cfu/mL) in triplicates and incubated in incubator Shaker at 28± 2 °C and 180 rpm for 4 days in dark. The bacterial cultures were centrifuged at 6,000 rpm for 30 min. Estimation of indole-3-acetic acid (IAA) in the supernatants was done using colorimetric assay (Brick et al., 1991). From a standard curve prepared with known concentration of IAA, the quantity in the culture was determined and expressed as μg/mL. Hydrogen cyanide production Screening of bacterial isolates for hydrogen cyanide (HCN) production was carried out following the method described by Bakker and Schippers (1987). Bacterial cultures were streaked on nutrient agar medium containing 4.4 g/L of glycine. A Whatman filter paper No. 1 soaked in 0.5% picric acid solution (in 2% sodium carbonate) was placed inside the plate lid. Plates were sealed with parafilm and incubated at 28±2 °C for 4 days. HCN production was evaluated according to the colour change, ranging from yellow to orange. Siderophore production Siderophore production by rhizobacterial isolates was detected according to Schwyn and Neilands (1987). Autoclaved CAS agar medium was poured in each Petri dish. The bacterial inoculum was placed in the centre of a Petri dish. The plates were incubated in the dark at 28± 2 °C for 5 days. The change of CAS agar colour from blue to orange around bacterial colony was considered as positive production of siderophore. Nitrogen fixing activity The visual detection of nitrogen-fixing activities of the selected isolates were observed by using nitrogen-free malate-mannitol medium (NF-MM) (Herman et al., 1994), containing bromothymol blue (BTB) as an indicator. The medium was inoculated by the bacterial isolates and incubated at 28± 2 °C for 24 h. The blue coloured zone producing isolates were marked as nitrogen fixers in the solid culture conditions. The colouring zone was calculated by deducting the colony diameter from the colouring zone diameter (Dobereiner and Day, 1976). Ammonia production All the bacterial isolates were tested for the production of ammonia as described by Cappuccino and Sherman (1992). Overnight grown bacterial cultures were inoculated in 10 mL of peptone broth and incubated at 28± 2 °C for 48 h on a shaker. After incubation 0.5 mL of Mal. J. Microbiol. Vol 13(2) June 2017, pp. 124-131 126 ISSN (print): 1823-8262, ISSN (online): 2231-7538 Nessler’s reagent was added. The development of faint yellow to dark brown colour indicated the production of ammonia. Biochemical identification of bacterial isolates The bacterial isolates were further characterized by Gram staining, catalase, oxidase, starch hydrolysis activity and motility followed by biochemical identification using API 20NE kit (BioMérieux, France). API 20 NE is a standardized system for the identification of non-fastidious, non-enteric Gram-negative rods combining 8 conventional tests, 12 assimilation tests. The results were interpreted with the API WebTM software (version 7.0). Statistical analysis The data obtained in this study was subjected to analysis of variance (ANOVA) and comparisons of means were performed by Newman and Keuls test at p ≤ 0.05 using statbox. The correlation between phosphate solubilization and pH was explored by Excel. RESULTS The pH of soil samples ranged from 7.82 to 8.46, moisture content from 10.52 to 18.6% and electrical conductivity from 6.1 to 13.3 ds/m. A total of 104 phosphate solubilizing isolates were obtained from different saline soil sites at Relizane (western Algeria). Out of these isolates 41 were selected for their phosphate solubilizing activity. These were screened for their plant growth promoting activities such as indole acetic acid (IAA), Siderophore production, HCN production, nitrogen fixing ability and ammonia production. Phosphate solubilization ability is marked by the formation of transparent halos around bacterial colony on solid Pikovskaya medium supplemented with tricalcium phosphate (Ca3(PO4)2) as a only source of phosphorus (Figure 1). All the 104 isolates were able to produce transparent halo around the colony with different diameter which indicates a positive phosphate solubilisation ability and they were also capable to solubilize inorganic phosphorus (Ca3(PO4)2) in liquid medium. The results showed that the phosphate solubilizing ability in NBRIP liquid medium of test isolates varied from 24.29 to 738.57 μg/mL using TCP as a source of insoluble P (data of all PSB are not given). The PSB showing the highest amount of solubilized phosphorus are chosen to be tested for their plant growth promoting traits (41 isolates). The isolate that showed the best capability in solubilizing phosphate was PSB91 (738.57μg/mL). pH values of the bacterial cultures decreased from initial value of 7.0 to values ranged from 3.9 to 4.61. A highly significant negative (r = -0, 86) correlation was observed between the amounts of solubilized P and pH values (Table 1). Figure 1: Phosphate solubilisation by bacterial isolate. Table 1: Phosphate solubilization by selected bacterial isolates. Isolate Concentration of phosphate (μg/ml) Final pH of P solubilization PSB3 605.34±15.91d 4.11±0.15 PSB5 525.60±14.73j 4.35±0.26 PSB8 652.83±25.28b 3.90±0.56 PSB11 515.90±12.78j 4.38±0.89 PSB13 584.17±13.78e 4.27±0.21 PSB16 553.62±24.17h 4.32±0.72 PSB18 555.86±16.60gh 4.31±0.41 PSB22 642.49±22.74c 3.92±0.46 PSB24 587.05±19.65e 4.18±0.27 PSB27 566.17±17.14fg 3.97±0.82 PSB29 562.90±21.92fgh 4.18±0.63 PSB31 410.89±16.62p 4.35±0.51 PSB34 453.28±16.95n 4.26±0.32 PSB36 564.74±11.5fg 4.26±0.65 PSB38 402.58±15.71pq 4.47±0.12 PSB41 610.96±13.87d 4.12±0.43 PSB43 571.22±18.70f 4.22±0.74 PSB44 541.59±23.67i 4.32±0.75 PSB45 601.85±22.60d 4.11±0.95 PSB49 561.33±12.03fgh 4.35±0.31 PSB52 370.04±10.61r 4.53±0.45 PSB56 570.63±16.51f 4.22±0.15 PSB58 398.00±11.76q 4.57±0.61 PSB64 487.71±15.35l 4.44±0.34 PSB66 502.25±14.83k 4.46±0.85 PSB67 481.71±16.49l 4.42±0.22 PSB70 568.05±25.89f 4.25±0.12 PSB71 447.20±17.94n 4.33±0.41 PSB72 466.94±18.60m 4.34±0.33 PSB74 435.52±20.87o 4.50±0.23 PSB78 348.05±15.24s 4.55±0.29 PSB82 327.50±16.50t 4.61±0.57 PSB85 398.70±12.42q 4.52±0.45 PSB87 538.37±12.70i 4.23±0.11 PSB89 521.9±14.66j 4.30±0.15 PSB91 738.56±16.07a 4.01±0.77 PSB93 455.13±13.18n 4.35±0.67 PSB95 410.35±22.16p 4.42±0.81 PSB97 521.77±23.66j 4.27±0.36 PSB99 407.67±15.57pq 4.47± 0.37 PSB102 604.37±17.85d 4.14±0.41 r -0.86 Mal. J. Microbiol. Vol 13(2) June 2017, pp. 124-131 127 ISSN (print): 1823-8262, ISSN (online): 2231-7538 Phosphate solubilizing bacteria were assayed for their capacity to produce IAA in LB medium supplemented with L-tryptophan as precursor (1%) by the appearance of pink colour after addition of Salkowski reagent to the culture (Figure 2). IAA production was revealed by almost all the bacterial isolates and IAA quantities of estimated ranged from 10.90 to 63.35 μg/mL as shown in Table 2. A relatively higher content of IAA was found in the culture of bacterial isolate PSB45 followed by PSB85 and PSB91 with 37.43 and 32.85 μg/mL respectively compared to other isolates. Figure 2: IAA production by bacterial isolates. Among the 41 isolates, only twelve between them were recorded positive for HCN production by changing filter paper from yellow to orange (Table 2, Figure 3). The ability of the tested bacterial isolates to produce siderophores in vitro was assessed qualitatively using the CAS-agar plate assay. Of the 41 bacterial isolates tested, 32 strains produced siderophores. Siderophore production capacity of the isolates was evaluated according to the diameter of the orange halo around bacterial colonies as shown in Figure 5, as weak (4-10 mm), moderate (11- 20 mm) and strong (higher than 21mm). Amongst the siderophore producing isolates, 18 were weak siderophore producers, 10 isolates were moderate siderophore producers while 7 isolates were strong siderophore producers (Table 2). Figure 3: HCN production by isolate. Phosphate solubilizing bacteria were also tested for their ability to fix nitrogen on nitrogen free medium (NF-MM) supplemented by BTB used as colour indicator to detect the release of ammonium in the culture as shown in Figure 5. Among the 41 bacterial isolates 31 were able to fix nitrogen by changing medium colour from green to blue colour. The diameter of coloration zone ranged from 7 to 26 mm. The highest value of diameter of coloration zone was obtained with the isolate PSB41 (Table 2). All our selected isolates were capable to produce ammonia (Table 2). Table 2: Characterization of bacterial isolates for multiple plant growth promoting traits. Isolate Concentration of IAA (μg/ml) HCN production SDR production Zone diameter of N fixation Ammonia production PSB3 13.80±2.63p + +++ 17 + PSB5 14.37±3.15n - - 14 + PSB8 14.84±1.12l - - 16 + PSB11 18.05±3.83g - + 11 + PSB13 11.59±4.35vw - + 09 + PSB16 12.62±3.16s - + 13 + PSB18 17.54±5.10h - ++ 12 + PSB22 11.57±2.36vw - + - + PSB24 11.48±1.94w - - 07 + PSB27 11.56±3.57vw - - 12 + PSB29 10.90±4.67z + +++ 11 + PSB31 10.77±2.45z - - 11 + PSB34 11.91±3.65u - + 10 + PSB36 16.90±3.05i + + - + PSB38 14.74±2.05m - - - + PSB41 22.67±3.55d - + 26 + PSB43 12.36±3.69t + +++ 25 + PSB44 22.73±2.54cd + + 11 + PSB45 63.35±5.28a + ++ 07 + PSB49 11.95±3.54u - + 12 + PSB52 11.66±3.45v - + - + PSB56 11.22±2.66x - + - + PSB58 13.60±3.67q - + 21 + PSB64 13.84±3.54p - + 18 + PSB66 15.49±3.05k + ++ 17 + PSB67 22.81±7.69c - + 10 + PSB70 20.21±6.76f - - 16 + PSB71 18.05±4.11g - ++ 17 + PSB72 15.45±3.84k - + - + PSB74 14.02±3.58o + ++ 18 + PSB78 20.60±5.84e + ++ 13 + PSB82 12.95±4.64r + +++ 22 + PSB85 37.43±2.57a - + 14 + PSB87 27.26±3.38ab - + 11 + PSB89 27.10±1.99b - - - + PSB91 32.85±2.32a - - - + PSB93 16.08±4.56j - ++ 07 + PSB95 16.12±4.03j + ++ 09 + PSB97 12.31±5.54t + ++ - + PSB99 11.05±2.69y - ++ 13 + PSB102 12.64±2.76s - + - + +, positive; ++, moderately positive; +++, strongly positive; -, negative A total of forty one isolates of rhizobacteria obtained were biochemically identified by several tests. 20 bacterial isolates were identified as genus Pseudomonas which is the most dominant isolate found including Gram-negative, rod shaped Pseudomonas luteola and Pseudomonas fluorescens, followed by Aeromomas hydrophila (10 isolates). Some of these are Gram-positive spore forming bacteria identified as Bacillus sp. (8 isolates). Only 3 isolates were Gram-negative and rod shaped identified as B. cepacia (Table 3). Mal. J. Microbiol. Vol 13(2) June 2017, pp. 124-131 128 ISSN (print): 1823-8262, ISSN (online): 2231-7538 Figure 4: Siderophore production by bacteria. In this study the highest amounts of released phosphorus were solubilized by Pseudomonas with 24.39% followed by Bacillus sp. (12.19%), Aeromomas (9.75%) and finally Burkholderia with 0.41%. Bacteria producing the highest concentration of IAA were isolates belonging to the genus Pseudomonas (24.39%) followed by Aeromonas (9.75%), Bacillus (8.33%) and Burkholderia with 0.41%. For HCN production Aeromonas was the higher producer (14.63%) followed by Pseudomonas (12.19%) Burkholderia (0.41%), and finally Bacillus with 0%. Isolates belonging to Pseudomonas were able to produce siderophore with 48. 78% followed by Aeromonas (14.63 %), Burkholderia (7.31 %) and Bacillus with 0%. The majority of isolates belonging to genus Pseudomonas were able to fix nitrogen with Table 3: Identification of selected bacterial isolates. Isolate Gram’s staining Shape Catalase Oxydase Amylase Motility ID PSB3 - rod + - + + P. luteola PSB5 + rod + - + + Bacillus sp. PSB8 + rod + + + + Bacillus sp. PSB11 - coccibacilli + - - + A. hydrophila PSB13 - rod + - + + P. fluorescens PSB16 - rod + - + + P. luteola PSB18 - rod + - - + P. luteola PSB22 - rod + + + + B. cepacia PSB24 + rod + - + + Bacillus sp. PSB27 + rod + + + + Bacillus sp. PSB29 - coccibacilli + + - + A. hydrophila PSB31 + rod + - + + Bacillus sp. PSB34 - rod + - + + P. luteola PSB36 - rod + - + + P. luteola PSB38 + rod + + + + Bacillus sp. PSB41 - rod + - + + P. luteola PSB43 - coccibacilli + + - + A. hydrophila PSB44 - coccibacilli + + - + A.hydrophila PSB45 - rod + - + + P. luteola PSB49 - rod + - + + P. luteola PSB52 - rod + - + + P. luteola PSB56 - rod + - + + P. luteola PSB58 - rod + - + + P. luteola PSB64 - rod + - + + B. cepacia PSB66 - rod + + + + P. fluorescens PSB67 - rod + - + + P. luteola PSB70 + rod + - + + Bacillus sp. PSB71 - rod + - + + P. luteola PSB72 - coccibacilli + + - + A. hydrophila PSB74 - Rod + + + + B. cepacia PSB78 - coccibacilli + + - + A.hydrophila PSB82 - rod + + - + A.hydrophila PSB85 - rod + - + + P. luteola PSB87 - rod + - + + P. luteola PSB89 - coccibacilli + + - + A. hydrophila PSB91 + Rod + - + + Bacillus sp. PSB93 - rod + - + + P. luteola PSB95 - rod + - + + P. fluorescens PSB97 - coccibacilli + + - + A. hydrophila PSB99 - rod + + - + A.hydrophila PSB102 - rod + - + + P. luteola Mal. J. Microbiol. Vol 13(2) June 2017, pp. 124-131 129 ISSN (print): 1823-8262, ISSN (online): 2231-7538 19.51% followed by Aeromonas and Bacillus (19.51% and 12.19% respectively) and finally Burkholderia with 4.87% Figure 5: Nitrogen fixation by bacterial isolates. DISCUSSION Plant growth promoting rhizobacteria (PGPR) are a group containing a large number of bacteria number found in the rhizosphere, at root exteriors and in association with them, which can enhance directly and/or indirectly yield and growth of agricultural crops. The capability of some rhizospheric microorganisms to solubilize soil phosphate and make it available for plants by converting it into accessible forms like orthophosphate, is an important activity in a PGPB for improving plant growth (Chen et al., 2006). In our study 104 phosphate solubilizing bacterial isolates were isolated from wheat plant rhizosphere of salt affected soil; these bacteria are more effective in phosphorus solubilization than fungi as reported by Alam et al. (2002). Our rhizobacterial isolates were biochemically identified as Pseudomonas, A. hydrophila, Bacillus sp. and B. cepacia. Srinivasan et al. (2012) also isolated Pseudomonas and Bacillus as phosphate solubilizers from salt affected soil. Moreover A. hydrophila and Bacillus sp. were isolated from the rhizospheric soil of wheat grown in saline soil by Ashraf et al. (2004). In quantitative estimation, all isolates showed diverse levels of phosphate solubilizing activity. The higher concentration of released phosphorus in cultures was exhibited by genus Pseudomonas followed by Bacillus. The same results were described by Ahmad et al. (2008) who reported that solubilization of phosphate was commonly detected in the isolates of Bacillus and Pseudomonas. Prior reports defined some Burkholderia strains as competent phosphate solubilizers (Peix et al., 2001; Caballero-Mellado et al., 2007). pH of bacterial cultures dropped significantly compared to the control. Similar results were observed by Mardad et al. (2013) and Alam et al. (2002). A negative correlation (r = −0.86) was detected between the amounts of solubilized phosphorus of bacterial cultures and their pH values. The same negative correlation was reported by Xiao et al. (2009). Mainly, the mode of action of phosphate solubilising bacteria in soil is by the secreting of acids into the medium (Khan et al., 2014; Oteino et al., 2015). Hence, various genus of bacteria including Bacillus, Pseudomonas, Enterobacter, Serratia, and Azotobacter sp. employ many solubilization reactions, such as acidification, exchange reactions, chelation and production of acids to release phosphorus from mineral complexes (Pandey and Maheshwari, 2007). Recently many researchers have studied production of phytohormones by PGPR (Rajkumar and Freitas, 2008; Ahmad and Khan, 2012). The most important plant growth regulators produced by phosphate solubilizing microorganisms are auxins (Oves et al., 2013). 75% of bacteria isolated from saline soil have the capacity to produce IAA which concluded that saline soil is a rich source of IAA producing bacteria. A high level of IAA production by Pseudomonas was noted in our study and by other researchers (Ahmad et al., 2008; Kumar et al., 2012). Other IAA producing bacteria belongs to Aeromonas (Halda-Alija, 2003) and Bacillus (Swain et al., 2007) were also reported. HCN is a volatile, secondary metabolite that suppresses the development of microorganisms. An important role of HCN produced by bacteria from rhizosphere in biological control of pathogens has been described (Siddiqui et al., 2006). In the current study isolates belonging to genus of Aeromonas, Burkholderia and Pseudomonas were able to produce HCN. To date various bacterial genera are able to produce HCN, including Pseudomonas, Aeromonas, Alcaligenes and Rhizobium (Devi et al., 2007; Ahmad et al., 2008). Siderophores produced by PGPR can promote plant growth either directly or indirectly, using radiolabeled ferric siderophores (Sujatha and Ammani, 2013). Our results showed that isolates belonging to genus Pseudomonas and Burkholderia were the best siderophore producers but isolates belonging to Bacillus sp. were unable to produce them. Also García-Gutiérrez et al. (2012) found that all Pseudomonas strains isolated from soil were able to produce siderophore, while only one strain among Bacillus was able to produce such compounds. Luvizotto et al. (2010) reported that B. cepacia exhibit a high levels of siderophore production. The presence of nitrogen-fixing bacteria in soil along with its isolation and conversion into PGPR biofertilizer is an important strategy reducing the use of expensive chemical fertilizers especially in nutrient poor and degraded soils (Cakmakci et al., 2006). The majority of our rhizobacterial isolates have the ability to fix nitrogen. Currently there are evidences that plant stimulation effect by PGPR such as Azoarcus sp., Burkholderia sp., Gluconacetobacter is related to their ability to fix nitrogen (Vessey, 2003). In the study of Cakmakci et al, (2006), the quality and yield of wheat, spinach and sugar beet was improved using nitrogen fixing bacterial isolates Pseudomonas RC06 and Bacillus OSU-142 as biofertilizers. Zhang et al. (1996) reported that A. hydrophila have the ability to fix atmospheric nitrogen. Ammonia production is an essential trait exhibited by PGPR, which can effect indirectly plants growth (Yadav et al., 2010). All our isolates were able to produce ammonia. These results are similar with those of Ahmed et al. (2008) Mal. J. Microbiol. Vol 13(2) June 2017, pp. 124-131 130 ISSN (print): 1823-8262, ISSN (online): 2231-7538 who revealed the production of ammonia commonly detected in all the isolates of Pseudomonas, Bacillus and Azotobacter. Similarly, all bacteria identified as Bacillus and Pseudomonas isolated by Yadav et al. (2010) from chickpea rhizosphere in India have the ability to produce ammonia. CONCLUSION Phosphate solubilizing bacteria isolated from wheat rhizosphere from saline soil located in western region of Algeria showed a high potential to produce growth promoting traits. Isolates belonging to the Bacillus and Pseudomonas showed a high phosphate solubilisation. Pseudomonas sp. was the highest producer of IAA and siderophore and had the capacity to fix nitrogen. A. hydrophila and B. cepacia showed high potential produce HCN and other PGP traits also. 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Effect of plant growth promoting rhizobacteria on seed germination and plant growth chickpea (Cicerarietinum L.) under in vitro conditions. Biological Forum An International Journal 2(2), 15-18. Zhang, F., Dashti, N., Hynes, R. K. and Smith, D. L. (1996). Plant growth promoting rhizobacteria and soybean [Glycine max (L.) Merr.] nodulation and nitrogen fixation at suboptimal root zone temperatures. Annals of Botany 77, 453-459., 2017-11-02)Résumé Les bactéries solubilisant le phosphate jouent un rôle vital dans la fertilité du sol et par conséquent favorisent la croissance des plantes. L’objectif globale de cette étude consiste à isoler et identifier des ...
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