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Ann Microbiol (2017) 67:801–811 https://doi.org/10.1007/s13213-017-1308-9 ORIGINAL ARTICLE Characterization of phosphate-solubilizing bacteria exhibiting the potential for growth promotion and phosphorus nutrition improvement in maize (Zea mays L.) in calcareous soils of Sinaloa, Mexico 1 1 1 Jesús A. Ibarra-Galeana & Claudia Castro-Martínez & Rosario A. Fierro-Coronado & 1 1 Adolfo D. Armenta-Bojórquez & Ignacio E. Maldonado-Mendoza Received: 27 April 2017 /Accepted: 18 October 2017 /Published online: 2 November 2017 Springer-Verlag GmbH Germany and the University of Milan 2017 Abstract Greenhouse bioassays were used to examine the B. megaterium may improve P nutrition and growth to a level ability of selected strains of the rhizobacteria Sinorhizobium previously attained by the addition of soluble P-fertilizer at meliloti, Bacillus flexus and B. megaterium to solubilize phos- 40 w/v P. A non-sterile experiment showed a beneficial re- phorus (P) and to affect growth promotion and phosphorus sponse with B. megaterium but not with B. flexus.Wepropose nutrition in maize. These bacterial strains were found to de- utilizing these bacteria in P-deficient alkaline soils in future crease the pH and solubilize some forms of insoluble P, such field trials in order to evaluate their potential as biofertilizers. as tricalcium phosphate and hydroxyapatite, as well as to ex- hibit acid and alkaline phosphatase enzymatic activities in Keywords Phosphate-solubilizing bacteria Growth culture medium, properties that are possibly involved in P . . promotion Phosphorus nutrition Maize solubilization. Inoculation of the strains separately and as a consortium of the three bacteria (S. meliloti, B. flexus and B. megaterium) in P-deficient soil (4.33 w/v P) fertilized without Introduction P improved plant height, shoot and root dry weight, as well as P nutrition in the maize plants. Use of the B. flexus and B. Maize (Zea mays L.), an integral part of the Mexican culture megaterium strains separately and in a consortium positively and diet since antiquity, is the most important cereal crop in affected several growth parameters and P nutrition in plants Mexico.Overhalfofthe country’s cultivated surface is dedi- supplemented with insoluble P. No effect was observed when cated to maize cultivation, with Sinaloa state leading the coun- pots in which the seedlings were growing were supplied with try in maize production (SIAP-SAGARPA 2016). A negative soluble fertilizer. A second assay using a P-deficient soil consequence of this intense cultivation practice is that high (6.64 w/v P) showed that inoculation with the consortium of yields in maize production have required intensive phosphorus B. flexus and B. megaterium significantly increased growth (P) fertilization (Tilman et al. 2002;Harvey et al. 2009). This and total P content in maize plants. A dose–response P fertil- has in turn contributed to the nutrient enrichment of water bod- ization experiment using sterile P-deficient soil led us to con- ies, causing eutrophication and toxic algal blooms (Smith and clude that inoculation to soil of the mixture of B. flexus and Schindler 2009). Intensive P fertilization regimes also affect microbial diversity, leading to the loss of soil fertility and, con- Electronic supplementary material The online version of this article sequently, a decrease in crop yield. Finally, estimates indicate (https://doi.org/10.1007/s13213-017-1308-9) contains supplementary that high-quality P sources are becoming increasingly limited material, which is available to authorized users. (Gilbert 2009), while low-quality rock P is widely available. Plants can only utilize 20–30% of the P in phosphate fer- * Ignacio E. Maldonado-Mendoza tilizers applied to agricultural soils due to the reactivity of P email@example.com with other cations (such as calcium in calcareous soils), which 1 causes the rapid mineralization and insolubilization of P in the Departamento de Biotecnología Agrícola, CIIDIR-Sinaloa, Instituto soil (Bashan et al. 2013). According to the soil taxonomy of Politécnico Nacional, Boulevard Juan de Dios Bátiz Paredes No. 250, CP 81101 Guasave, Sinaloa, Mexico the Food and Agriculture Organization of the United Nations 802 Ann Microbiol (2017) 67:801–811 (FAO) classification, the soil types in Northern Sinaloa soil are Materials and methods vertisol, feozem and cambisol, and they are characterized by an alkaline pH ranging from 7.5 to 8.4 (Ramírez Soto et al. Microorganisms 2010). This results in the mineralization of P in insoluble compounds such as dicalcium or tricalcium phosphate (TCP) Bacillus flexus, B. megaterium and Sinorhizobium meliloti and hydroxyapatite (Bashan et al. 2013;Shenet al. 2011). strains were used. Bacterial isolates were obtained from maize These problems linked to the use of P fertilizers have pro- rhizospheric soils of northern Sinaloa, Mexico. Strains were moted the search for environmentally friendly alternative identified based on their 16S rDNA gene sequence, as report- strategies that can improve crop production in low-P or P- ed in Figueroa-López et al. (2016). The Bacillus strains were deficient soils. Applying microbial inoculants or biofertilizers obtained from microorganisms maintained in a preliminary that have P-solubilizing activities to agricultural soils is con- collection (CIIDIR-003; CIIDIR-Sinaloa, Mexico) and were sidered to be an environmentally friendly alternative that can stored at −70 °C in Luria Bertani (LB) broth with glycerol avoid/decrease the use of conventional P fertilizers (Sharma (15%, v/v). The Sinorhizobium strain was obtained from a et al. 2013; Zaidi et al. 2009). However, a P source is still collection that was specifically made to select for bacteria that needed for these bacteria to function, and this source can be can solubilize TCP [Ca (PO ) ] on solid medium (Fierro- 3 4 2 low-grade rock phosphate. Coronado, unpublished results) as a first rough indicator of One such alternative approach for sustainable agriculture potential P solubilization. In this work, the S. meliloti strain could use phosphate-solubilizing bacteria (PSB) to satisfy the was molecularly identified based on 16S rDNA gene sequenc- P requirements for plant growth (Lavakush et al. 2014; ing (GenBank accession number KU230303). Strains were −1 Richardson et al. 2011). The mechanisms used by PSB to stored at −70 °C in peptone–yeast (PY) medium [5 g L −1 −1 convert P-insoluble forms into available P-soluble forms in- peptone and 3 g L yeast extract with 10 ml L CaCl volve acidification, chelation, oxidation-reduction reactions (0.7 M sterile solution added after sterilizing the culture me- and secretion of strong organic acids (Bashan et al. 2013; dium)] with glycerol (15%, v/v). The three strains (S. meliloti, Chen et al. 2006; Young et al. 2013), as well as the synthesis B. flexus and B. megaterium) were tested for their ability to of several phosphatase enzymes (Richardson 2001). The ap- solubilize TCP using the qualitative P-solubilization plate plication of PSB to the soil can improve the availability of soil method as reported by Figueroa-López et al. (2016). After P, the absorption of nutrients (N and K) and root development testing the ability of the strains to solubilize TCP solubiliza- (Lopez-Arredondo et al. 2014). PSB application may also tion in solid medium, their ability to solubilize hydroxyapatite enhance plant growth through other mechanisms, such as was tested in liquid culture, as described in section Psolubi- symbiotic nitrogen fixation, aminocyclopropane-1- lization in liquid culture. carboxylate deaminase activity, growth control of phytopath- ogenic microorganisms and the production of ammonia, Phosphate solubilization in solid medium siderophores and phytohormones (Bashan and de-Bashan 2010; Pereira and Castro 2014; Vessey 2003). Solid Pikovskaya medium (per liter: 10 g glucose; 0.5 g yeast Sinorhizobium meliloti, Bacillus flexus and B. megaterium extract; 5gTCP;0.5gMgCl ·6H O; 0.2gKCl; 0.2gNaCl ; 2 2 2 are native maize rhizospheric bacterial isolates from northern 0.1gMgSO ·7H O; 15 g bacteriological agar) was used to 4 2 Sinaloa, Mexico. In a recent study these Bacillus strains were qualitatively assess the ability of bacterial isolates to solubilize observed to exhibit antagonistic activity against Fusarium phosphate. The final pH was adjusted to 7.00 ± 0.02. The verticillioides (Fv) in vitro (Figueroa-López et al. 2016). In strains were inoculated onto the solid agar plates and the plates an earlier study, the application of B. megaterium B5 in field incubated at 30 °C for 10 days. The experiment was trials demonstrated that this strain is capable of reducing the performed twice using three plates each time. Phosphate incidence (by 75.1%) and severity (by 30%) of maize ear rot, solubilization was assessed by measuring the clear zone in addition to increasing grain yield (Lizárraga-Sánchez et al. (area of P solubilization) surrounding each bacterial colony. 2015). Nevertheless, these strains must still be tested in green- The phosphate solubilization index was calculated using the house and field trials to demonstrate their ability to perform as formula: (colony diameter + halo zone diameter)/colony P solubilizers. The aim of this study was to characterize the diameter. phosphate solubilization of these strains in greenhouse bioas- says and to evaluate their potential as biofertilizers for use in P solubilization in liquid culture the promotion of growth and improvement of P nutrition in maize plants growing in P-deficient soil. Our hypothesis was Quantitative analysis of P solubilization was performed that when bacterial strains possessing the ability to solubilize using 250-mL Erlenmeyer flasks containing 50 mL of phosphate are tested under greenhouse conditions they should Pikovskaya medium. Final bacterial cell suspension con- improve P nutrition in and growth of maize plants. centrations were adjusted to a cell density of approximately Ann Microbiol (2017) 67:801–811 803 7 −1 3.0 × 10 CFU mL . Three flasks containing Pikovskaya (NPK 12/61/00); (3) fertilized with insoluble phosphate medium were inoculated with each presumptive isolate (TCP). This design also included five types of inoculation: 7 −1 (100 μLinoculum with 3.0 ×10 CFU mL ) and incubated (1) control without bacteria and (2) inoculated treatments with at 30 °C on a rotary shaker (200 rpm); pH and soluble P were S. meliloti, B. flexus, B. megaterium, and a consortium of measured at different time intervals (0, 1, 2, 3, 5, 7 and S. meliloti, B. flexus and B. megaterium, respectively. 10 days). Experiments were conducted twice per each isolate. Commercial white maize hybrid seeds (DeKalb DK-2038; The bacterial strains were assessed separately with two dif- Guasave, Mexico) were disinfected using a hydrothermal ferent sources of insoluble phosphate: TCP and hydroxy- treatment. Seeds were immersed in a Tween-20 solution (five apatite [Ca (PO ) (OH)]. After the predefined incubation drops of Tween 20 per 100 mL of sterile distilled water) and 5 4 3 period, the cultures were harvested by centrifugation at sonicated for 5 min. The Tween solution was then decanted 2300 g for 15 min. Sterile, non-inoculated medium served and the seeds immersed in a 0.75% sodium hypochlorite so- as the control. The amount of soluble inorganic phosphate lution and placed in a thermo-bath at 52 °C for 20 min. Finally, (Pi) remaining in the culture supernatant was measured the seeds were washed three times in sterile distilled water and using the phosphomolybdate blue colorimetric method allowed to air-dry in a laminar flow hood (Leyva-Madrigal (Murphy and Riley 1962). The amount of released Pi was et al. 2015). For seed inoculation, Bacillus strains were grown calculated based on a standard KH PO curve (Sigma- in LB medium, whereas S. meliloti was grown in PY broth. 2 4 Aldrich; St. Louis, MO). Sample pH was measured in the Cells in the exponential phase of growth were harvested by bacterial supernatant using a pH meter (model 223; Hanna centrifugation at 1000 g for 5 min. Bacterial inoculum was Instruments; Woonsocket, RI). prepared by re-suspending pelleted cells in either LB or PY broth for B. flexus and B. megaterium strains and S. meliloti, Phosphatase activity assays respectively. The rhizobacteria inoculum density was adjusted 8 −1 to 2.0 × 10 CFU mL . Seeds were pre-germinated for 5 days Acid and alkaline phosphatase activities of the bacterial in petri dishes with water-agar, and seeds showing contami- isolates were determined using a modified assay by Juma nation even after disinfection were discarded (< 3%). and Tabatabai (1988). Briefly, 100 μL of culture superna- Germinated seeds were immersed for 1 h in each of the bac- tant obtained by centrifugation of the bacterial cultures terial suspensions (S. meliloti, B. flexus, B. megaterium)or a (2300 g,15min)was incubatedat37°Cwith100 μLof mix of the three strains, adjusted to a final concentration of 8 −1 25 mM p-nitrophenyl phosphate and 400 μL of modified 2.0 × 10 CFU mL ). The seeds were then planted in pots universal buffer pH 5 (Öhlinger et al. 1996). After 1 h, the containing 1 kg soil/pot. Bacterial suspensions (5 mL/ reaction was terminated by adding 100 μLCaCl (0.5 M) pot = 1 × 10 CFU) were also inoculated onto the surface and 400 μLCaCO (0.5 M). After incubation, the absor- of the soil and on top of the maize seeds at the time of bance was read at 410 nm, and the amount of product planting. Two seeds were planted per pot; later, at the time obtained in the reactions was determined by extrapolation of seedling emergence, one of the seedlings was removed using a p-nitrophenol (p-NP) calibration curve (μmol p-NP so that there was one seedling per pot. This (1 seedling/pot) −1 mL ). Each bacterial isolate was assayed enzymatically in was considered to be an experimental unit, and five repli- two independent experiments with three biological replicates cates per treatment were set up in a completely randomized (three flasks) after overnight incubation (16 h) at 30 °C and design for a total of 75 plants. This experiment was repeated 200 rpm. twice independently. Five days after maize seedling emer- gence, a second bacterial inoculation was performed, in Greenhouse pot assays which 5 mL of bacterial inoculum (1 × 10 CFU) was added per pot at the same concentration as used previously. Pots The soil used in the pot experiments was collected from a were placed in a controlled greenhouse (photoperiod 12 h, maize field in northern Sinaloa. Soil was milled, sieved temperature range 28–30 °C, relative humidity range 60– (<2 mm) and mixed with vermiculite to a 1:3 ratio (v/v) and 75%). Plants were watered twice per week with 150 mL sterilized three times by Tyndallization (121 °C for 60 min on distilled water in order to reach substrate field water capac- 3 consecutive days with subsequent drying at room tempera- ity. Depending on the P treatment scheme, different P fer- ture). After sterilization, the soil physico-chemical properties tilization regimes containing various fertilizers were ap- −1 were measured as: pH 7.8, conductivity 0.19 mmhos cm , plied to the pot substrates. Only N was applied in the treat- organic matter 0.95% and (in w/v) NO 250, P Olsen 4.33, K ment that did not receive any P fertilization, in the form of 547, Ca 7876, Mg 1507, Na 161, Fe 10.36, Cu 5.02, Zn 2.30 0.33 g of urea (NPK 46/00/00). In the treatment for insolu- and Mn 4.25. The greenhouse assay was based on a complete- ble phosphate fertilization, P was added in the form of TCP −1 ly randomized design with three phosphate treatments: (1) (0.215 g TCP kg of substrate), and N was supplemented control without P fertilization; (2) fertilized with soluble P in the form of urea, as described in the previous treatment. 804 Ann Microbiol (2017) 67:801–811 In the treatment with soluble phosphate fertilization, phospho-vanadomolybdate colorimetric method (Ryan 0.164gofanNPK fertilizer (12/61/00) and0.29gofurea et al. 2007). were added. Equivalence between both nutrients was main- tained in all treatments: N and P were supplemented to Statistical analysis reach 330 w/v of N and 160 w/v of P (soluble or insoluble). Data from greenhouse pot assays were subjected to two-way analysis of variance using SAS 9.0 (SAS Institute, Cary, NC). Greenhouse pot assay using different P fertilization doses Greenhouse pot assays displaying different rates of P fertiliza- tion data were analyzed using a generalized linear model. Following the evaluation of the greenhouse assay described in Fisher’s least significant difference test was used for the post the previous section, both Bacillus strains were selected based hoc comparison of means. Correlations were performed be- on their potential to promote growth and to increase P content tween different variables, and Pearson’s correlation coefficients in maize tissues; in contrast, S. meliloti was not selected for were determined using the Statgraphics Centurion XVI.I statis- further use. Therefore, for the greenhouse pot assay described tical package (Statpoint Technologies, Inc., Warrenton, VA). here, we designed a dose–response experiment that contained a control without bacteria, and treatments were inoculated with B. flexus and B. megaterium both separately and as a Results mixture. Fertilization doses were established at 0, 40, 80 and 120 w/v P. Substrate analysis for this experiment indicated the Qualitative and quantitative assessment of phosphate following physico-chemical properties: pH 7.8, conductivity solubilization −1 0.16 mmhos cm , organic matter 0.98% and (in w/v) NO 25, P Olsen 6.64, K 581, Ca 9018, Mg 998, Na 168, Fe 11.4, Cu The bacterial strains tested differed in their ability to solubilize 4.60, Zn 2.00 and Mn 5.5. A completely randomized design TCP, based on the formation of clear halos around colonies with four treatments and four plants per treatment was utilized. growing on Pikovskaya agar medium. Specifically, S. meliloti The amounts used to fertilize each pot in each fertilization presented a solubilization index of 3.05. In contrast, the treatment were as follows (per kg of substrate): treatment (1) Bacillus strains did not produce any clear zone surrounding 0 g P, 0.33 g urea; treatment (2) 0.040 g P, 0.032 g urea; the colonies on this medium, even though these bacteria have treatment (3) 0.080 g P, 0.031 g urea; treatment (4) 0.120 g previously been shown to dissolve TCP on Pikovskaya agar P and 0.30 g urea. N concentration was established at 330 w/v, (Figueroa-López et al. 2016). as in the first pot assay. NPK (12/61/00) was used as the P To verify that these strains had not lost their ability to sol- fertilizer, and urea (NPK 00/46/00) was used as the N fertiliz- ubilize TCP, we performed an experiment in liquid er. A sterile soil/vermiculite mix (1:3 ratio, v/v) was used for Pikovskaya medium. The amount of soluble Pi and changes this last experiment. At the end of the experiment, the avail- in pH were monitored for 10 days in Pikovskaya broth able P remaining in the substrate of all treatments was deter- (Fig. 1). Bacterial inoculation significantly increased TCP sol- mined according to the method described by Olsen et al. ubilization and caused a significant drop in pH by all strains. (1954). In a separate trial, the effect of bacterial inoculation The non-inoculated control remained at the initial pH, and no was evaluated in non-sterile substrate fertilized without P P solubilization was recorded. Sinorhizobium meliloti had sol- (0 g P, 0.33 g urea). All other assay conditions were as −1 ubilized up to 592.85 μgPmL medium at day 10 and de- described in preceding Materials and Methods subsections. creased the pH to 4.48. The maximum P solubilization shown Both experiments, the one performed under sterile conditions −1 by Bacillus strains was 207.24 μgmL , with a final pH of and the one performed under non-sterile substrate conditions, 4.85. This result indicates that the Bacillus strains had not lose were repeated twice independently. their ability to solubilize phosphate. Moreover, the results show a clear negative correlation between the soluble P con- Plant sampling and analysis centration and pH (S. meliloti, r = − 0.876; B. flexus, r = − 0.812; B. megaterium, r = − 0.760; P <0.05). Plants were harvested after 30 days and their roots thor- Both S. meliloti and B. megaterium were able to solubilize oughly washed in tap water and deionized water. Shoot and hydroxyapatite, which as a P source is more insoluble than root biomass and shoot height were then recorded. The dry TCP (Fig. 1). Furthermore, these strains demonstrated a neg- biomass of shoots and roots was determined after they ative correlation between hydroxyapatite solubilization and were oven dried at 70 °C for 72 h. Oven-dried tissues were pH reduction in the culture medium (S. meliloti, r = −0.774, finely ground, and tissue (0.5 g) was digested according to P <0.05; B. megaterium, r = −0.766, P <0.05).The B. flexus Kirkpatrick and Bishop (1971). The digested samples were strain was unable to solubilize hydroxyapatite in liquid medi- used to determine the total P concentration in shoots by the um, even when a decrease in pH occurred. Ann Microbiol (2017) 67:801–811 805 Fig. 1 Solubilization of different types of insoluble phosphate forms Changes in solubilized hydroxyapatite levels in liquid culture of the tested by the rhizobacteria strains Sinorhizobium meliloti, Bacillus flexus strains (c) and the accompanying changes in the pH of the and B. megaterium. a, b Changes in solubilized tricalcium phosphate hydroxyapatite-containing medium (d). Different letters above the bars [TCP; Ca (PO ) ] levels in liquid culture of the tested strains (a) and the indicate significant differences at P < 0.05 between treatments according 3 4 2 accompanying changes in the pH of the TCP-containing medium (b). c, d to the Tukey least significant difference test Phosphatase activity biomass (Fig. 3). No significant differences were observed in any of the analyzed parameters when maize plants were Alkaline and acid phosphatase activities for the two Bacillus fertilized with soluble P [Electronic Supplementary Material strains were detected in the culture medium (Fig. 2). Alkaline (ESM) Table S1]. This experiment was repeated and the re- phosphatase activity was lower than that of acid phosphatase sults found were as follows: in substrate without P fertiliza- in B. flexus and B. megaterium and required more time to tion, the presence of B. flexus and B. megaterium promoted reach its peak activity (144 vs. 48 h, respectively). At 48 h, growth and increased total P content in the plant tissue (ESM acid phosphatase activity was 2.4- to 3.0-fold higher than that Table S2). Bacterial inoculation with strains B. flexus and of alkaline phosphatase in all strains. Sinorhizobium meliloti B. megaterium of a substrate fertilized with insoluble P had showed significantly lower both phosphatase activities than a beneficial effect on plant height, shoot dry biomass and the Bacillus strains. shoot total P content of the parameters evaluated with respect to the inoculated control (ESM Table S3). The data shown in Influence of rhizobacteria inoculation on Z. mays growth ESM Table S4 confirmed that when maize plants growing in a and P nutritioningreenhouseassays P-deficient substrate were fertilized with soluble P, the addi- tion of these bacterial strains had no significant effect. In the P-deficient substrate (without no P fertilizer), all bacte- rial treatments significantly promoted plant height and root biomass, whereas treatments that included B. flexus increased Evaluation of inoculation by Bacillus strains biomass and total P shoot content relative to the control and the bacterial mixture on maize plants in pot assays (Fig. 3). Plant height and total P shoot content increased sig- using different fertilization doses nificantly upon fertilization with insoluble P, relative to the control, when plants were inoculated with B. flexus, Based on the results obtained in the previous greenhouse as- B. megaterium and the bacterial consortium (S. meliloti, B. say, we selected the Bacillus strains for further characteriza- flexus and B. megaterium); inoculation with B. megaterium tion of their potential to promote maize growth and improve P nutrition. B5 or the consortium also enhanced production of shoot 806 Ann Microbiol (2017) 67:801–811 Fig. 2 Phosphatase activities secreted into the culture medium by the rhizobacteria strains S. meliloti, B. flexus and B. megaterium. a, b Alkaline phosphatase (a) and acid phosphatase (b)activity[in μmol −1 −1 p-nitrophenol (p-NP) mL h ] in liquid culture. Sinorhizobium meliloti was cultured in PY broth; B. flexus and B. megaterium were cultured in LB medium. Different letters above the bars indicate a significant difference at P <0.05 between treatments according to the Tukey least significant difference test Substrate that had been fertilized in the presence of B. flexus Discussion or the bacterial mixture B. flexus and B. megaterium (without P) induced plant height, root and shoot biomass and shoot total In this study, we observed a negative correlation between pH P content to values similar to the control fertilized with 40 w/v and P solubility in liquid cultures of rhizobacteria. A similar P. At fertilization with 40 w/v P, the B. flexus and B. phenomenon was reported by Sridevi and Mallaiah (2009), megaterium mixture induced plant height to levels that were who demonstrated that one particular strain of Rhizobium sp. similar to those of the control fertilized with 80 w/v P. Shoot decreased the pH of the growth medium from 7 to 4.05 and dry biomass and total P content increased significantly in the increased P solubilization. Soluble P concentration values of −1 treatments that included B. megaterium relative to the non- 200 μgmL (final pH 4.46; Oliveira et al. (2009)and −1 inoculated control, although they did not reach the values ob- 96.73 μgmL (final pH 5.8; Yu et al. (2011) have been tained with 80 w/v P (Table 1). reported in liquid cultures of strains of Bacillus sp. In our Inoculation with the bacterial mixture increased available P study, TCP solubilization in cultures of the Bacillus strains for the substrate that received fertilization without any P, as was accompanied by an increase in the pH of the culture well as the substrate fertilized with 40 w/v P. Inoculation with medium at day 5 (B. flexus) and day 7 (B. megaterium). This B. flexus significantly increased available P, but only in the slight increase in pH could be due to a decrease in the me- non-fertilized substrate. Non-significant differences in the dium’s carbon source (glucose) due to bacterial growth, which substrates fertilized with 80 and 120 w/v P were observed, can inhibit the gene expression of metabolic pathways for relative to the non-inoculated control (Table 2). diverse carbon sources. This in turn causes a decrease in or- Bacterial inoculation employing a non-sterile substrate fer- ganic acid synthesis and its secretion into the medium. tilized without P demonstrated a beneficial effect of strain Secreted organic acids chelate insoluble P to make it soluble B. megaterium on plant height, shoot biomass and total P by decreasing the medium concentration and causing a slight content but no effect on root biomass with respect to the un- increase in pH (Marciano-Marra et al. 2012). A similar phe- treated control, while inoculation of the bacterial mixture nomenon has been reported for Penicillium spp. and (B. flexus and B. megaterium) increased only plant height Burkholderia cepacia (Nath et al. 2012;Zhao etal. 2014). and shoot biomass (Table 3). Bacillus flexus did not cause We demonstrated an inverse relationship between pH and P any significant change in any of the parameters evaluated with solubilization, a result in line with reports from Bianco and respect to the control. Defez (2010) and Collavino et al. (2010), suggesting that Ann Microbiol (2017) 67:801–811 807 Fig. 3 Effect of inoculation by the rhizobacteria strains S. meliloti, deviation of n = 5 trials). A two-way analysis of variance was performed B. flexus, B. megaterium and consortium (S. meliloti, B. flexus and to determine the influence of P treatments and bacterial inoculation. The B. megaterium) on the growth of maize plants and P nutrition. control was without bacteria; all other cultures contained S. meliloti, Greenhouse experiments were performed either in substrate without P B. flexus, B. megaterium, or a consortium of S. meliloti, B. flexus and fertilization or substrate fertilized with insoluble P. Parameters measured B. megaterium. Different letters above the bars indicate significant differ- were plant height (a), root dry biomass (b), shoot dry biomass (c)and ences at P < 0.05 between treatments according to Fisher’s least signifi- shoot total P content (d). Results are presented as the mean ± standard cant difference test medium acidification facilitates P solubilization. The secretion Phosphatase activity contributes to the solubilization of of organic acids by PSB plays a major role in soil P solubili- organic phosphates in the soil (Richardson 2001). Organic P zation by lowering the pH and enabling the replacement generally accounts for 30–65% of total soil P content, depend- 2+ 2+ 3+ 3+ (Ca ) and/or chelation (Ca ,Fe ,Al ) of the metal ions ing on soil type and land management (Richardson et al. that usually form insoluble P complexes (Bashan et al. 2013). 2009). Bacterial strains from our study have phosphatase ac- Phosphate solubilization by bacteria native to alkaline tivities similar to those reported in the literature. For example, −1 −1 soils, such as those from our region, has not only to be tested acid phosphatase activity can reach 8.0 μmol mL h in with TCP but also with other more insoluble forms of phos- Rhodococcus spp. strains (Pereira and Castro 2014), while phate such as hydroxyapatite, as suggested by Bashan et al. alkaline phosphatase activity was measured at 2.15 μmol −1 −1 (2013). Our findings from the testing of TCP and hydroxyap- mL h in Xanthomonas maltophila (De Freitas et al. atite as more insoluble forms of phosphate are coincident with 1997). Importantly, the de novo synthesis of these enzymes those reported by Bashan et al. (2013). We observed that our is stimulated when the level of inorganic P in the growth selected phosphate solubilizer bacterial strains possessed dif- medium is limiting (Dick et al. 2011), which explains why ferential hydroxyapatite solubilization ability. For example, phosphatase activity is determined in a medium without a strain B. flexus was unable to solubilize hydroxyapatite at phosphate source; otherwise, the presence of soluble P in- all, possibly due to the type of organic acids that B. flexus hibits acid and alkaline phosphatase activity (Hidayat et al. secretes into the medium. The solubilization efficiency of or- 2006; Kapri and Tewari 2010). ganic acids for chelating metal cations is strongly influenced Our results show that maize growth is promoted and P by the number of carboxyl and hydroxyl groups, as well as the nutrition is increased in cultures inoculated with the bacterial type and position of the ligand (Kpomblekou-a and Tabatabai strains when a P-deficient substrate without the addition of P 1994). fertilizer or with the addition of insoluble phosphate is used. 808 Ann Microbiol (2017) 67:801–811 Table 1 Effect of bacterial inoculation under different phosphorus fertilization treatments Treatments Plant height (cm) Root dry biomass (g) Shoot dry biomass (g) Shoot total P content (mg) 0w/v P+ B4 73.75 ± 7.97 f 0.54 ± 0.20 f 1.18 ± 0.32 e 1.93 ± 0.51 h 0w/v P+ B5 56.75 ± 2.99 g 0.35 ± 0.18 h 0.53 ± 0.11 f 0.61 ± 0.14 j 0w/v P+ B4-B5 78.00 ± 6.48 f 0.67 ± 0.27 fgh 1.17 ± 0.28 e 1.44 ± 0.28 hi 40 w/v P + B4 77.25 ± 2.87 f 0.55 ± 0.03 gh 1.17 ± 0.23 e 1.41 ± 0.30 hi 40 w/v P + B5 86.75 ± 6.99 e 0.93 ± 0.26 ef 2.51 ± 0.22 d 3.17 ± 0.25 g 40 w/v P + B4-B5 95.00 ± 4.55 d 0.87 ± 0.26 fg 2.13 ± 0.26 d 2.76 ± 0.31 g 80 w/v P + B4 101.75 ± 3.59 cd 1.30 ± 0.27 d 4.43 ± 0.31 c 5.91 ± 0.50 e 80 w/v P + B5 108.50 ± 5.26 abc 1.35 ± 0.28 d 4.91 ± 0.12 b 6.71 ± 0.33 d 80 w/v P + B4-B5 103.75 ± 4.11 bc 1.50 ± 0.23 cd 4.54 ± 0.27 bc 4.93 ± 0.38 f 120 w/v P + B4 110.25 ± 2.06 ab 1.86 ± 0.31 bc 5.76 ± 0.38 a 8.63 ± 1.18 a 120 w/v P + B5 110.75 ± 5.06 ab 2.04 ± 0.41 ab 6.02 ± 0.48 a 7.61 ± 0.64 bc 120 w/v P + B4-B5 111.25 ± 4.35 a 2.35 ± 0.27 a 6.06 ± 0.41 a 7.11 ± 0.67 cd Control 0 w/v P 60.25 ± 4.57 g 0.38 ± 0.23 h 0.65 ± 0.19 f 0.79 ± 0.2 ij Control 40 w/v P 73.50 ± 4.73 f 0.63 ± 0.17 fgh 1.14 ± 0.32 e 1.35 ± 0.31 hi Control 80 w/v P 101.75 ± 5.12 cd 1.28 ± 0.25 de 4.51 ± 0.26 bc 5.61 ± 0.54 ef Control 120 w/v P 105.00 ± 4.97 abc 2.09 ± 0.48 ab 5.75 ± 0.18 a 8.07 ± 0.54 ab Values are presented as the mean ± standard deviation (SD) (n = 4 trials). Different lowercase following the values indicate a significant between- treatment difference at P < 0.05 according to Fisher’s least significant difference test A generalized linear model was performed to determine the interaction between bacterial inoculation and phosphate treatment P, Phosphorus Control: treatment without bacteria. B4, B5, B4–B5: treatment with Bacillus flexus strain B4,with B. megaterium strain B5, with a mixture of strains B4 and B5,respectively These results are in agreement with those obtained using other microorganisms (Bago et al. 2000). Under these conditions, bacterial isolates in maize (Gurdeep and Reddy 2015; P is acquired through the direct absorption pathway via the Hameeda et al. 2008; Pereira and Castro 2014; Zahid 2015). root hairs (Nagy et al. 2009). Therefore, it is possible that the Inoculation with the S. meliloti strain did not present any sig- PSB–plant root association is limited in soils with high levels nificant effects on growth promotion or P nutrition in substrate of available phosphate. fertilized with an insoluble phosphate form. Maize inoculation with the bacterial strains improved P Maize fertilized with soluble P to attain a soil concentration nutrition and growth parameters at 0 and 40 w/v P when dif- of 80–120 w/v P or 160 w/v P (ESM Table S1) did not show ferent doses of P fertilization were used; this finding is con- any significant differences in growth or P nutrition when in- sistent with the use of the B. flexus and B. megaterium bacte- oculated with bacteria. A previous study using soils with high rial mixture. This beneficial effect of this synergistic bacterial levels of available P found that plants can bypass the energy mixture has previously been reported in maize (Pereira and cost required to establish an association with beneficial Castro (2014), wheat (Turan et al. (2012), walnut seedlings Table 2 Available phosphorus Bacterial treatment P fertilization treatment regimen remaining in the substrate after harvesting of plants grown in 0 w/v P 40 w/v P 80 w/v P 120 w/v P different phosphorus fertilization treatment regimens and with Control 6.06 ± 1.00 b 17.55 ± 1.1 7b 29.62 ± 1.70 a 33.50 ± 1.43 a different bacterial strain inoculations Bacillus flexus 7.77 ± 0.89 a 18.79 ± 1.11 ab 28.63 ± 0.97 a 32.02 ± 2.26 a B. megaterium 5.42 ± 1.06 b 17.42 ± 1.52 b 29.64 ± 1.02 a 32.82 ± 1.43 a B. flexus + B. megaterium 7.92 ± 0.59 a 19.59 ± 0.86 a 28.99 ± 0.96 a 34.41 ± 1.51 a Values are presented as the mean ± SD (n = 4 trials) One-way analysis of variance (ANOVA) was performed to determine the influence of different P fertilization treatments with bacterial inoculation. Different lowercase following the values indicate a significant between- treatment difference at P < 0.05 according to Fisher’s least significant difference test Ann Microbiol (2017) 67:801–811 809 Table 3 Effect of bacterial Bacterial treatment Plant height Root dry Shoot dry Shoot total P inoculation of substrate under (cm) biomass (g) biomass (g) content (mg) non-sterile conditions and when fertilized without phosphorus Control without bacterial 61.00 ± 4.24 b 0.67 ± 0.12 ab 0.86 ± 0.13 b 1.42 ± 0.32 b B. flexus 66.80 ± 5.32 ab 0.57 ± 0.19 b 0.88 ± 0.28 b 1.57 ± 0.46 b B. megaterium 71.00 ± 4.32 a 0.73 ± 0.17 ab 1.30 ± 0.23 a 2.53 ± 0.58 a Mixture of B. flexus and 69.80 ± 3.77 a 0.89 ± 0.18 a 1.22 ± 0.17 a 1.91 ± 0.36 ab B. megaterium Values are presented as the mean ± SD (n = 4 trials) One-way ANOVA was performed to determine the influence of bacterial inoculation on non-sterile substrate and fertilized without P. Different lowercase following the values indicate a significant between-treatment difference at P < 0.05 according to Fisher’s least significant difference test (Yu et al. (2011) and mangroves in Mexico (Rojas et al. 2001). positive effects on growth promotion and P nutrition in maize, Bioavailable P present in the substrate at the end of the bioas- suggest that they be tested in the field as a bacterial mixture to say indicates that B. flexus was able to increase the levels of alleviate P deficiencies in low-P soils found in regions of soluble P in the substrate without fertilization. Furthermore, Sinaloa where P fertilizers are not a feasible option for low- the B. flexus and B. megaterium mixture increased the P levels income farmers. We propose utilizing these two strains in in the treatment without any P fertilization, or when the sub- combination since a synergistic effect is, with one strain im- strate was fertilized with 40 w/v P, relative to the non- proving P nutrition and the other acting as a growth promoter. inoculated controls. Similar results have been found using The inoculation of this bacterial mixture (B. flexus and B. different bacterial consortia (Han and Lee 2006; Pereira and megaterium) in maize plants combined with an integrated Castro 2014). crop management approach in alkaline P-deficient soils could When the soil microbiota was tested in an experiment using be a viable alternative to improve crop productivity, increase a non-sterile substrate, we observed that the beneficial effects soil fertility and provide a sustainable strategy for the applica- of B. megaterium and of the bacterial mixture on P nutrition tion of phosphate fertilizers. and growth were not affected by competition or interaction with the native soil microorganisms; in contrast B. flexus did Acknowledgements This work was supported by the Secretaría de not behave well. Bacterial strains may show beneficial effects Investigación y Posgrado del Instituto Politécnico Nacional (SIP-IPN) (2014-2016). JAIG received M.Sc. fellowships from the Consejo under controlled conditions, but their behavior may differ Nacional de Ciencia y Tecnología (CONACyT) and the Programa de when applied to natural types of soils (Egamberdiyeva 2007; Becas de Estímulo Institucional de Formación de Investigadores (2014– De-Bashan et al. 2010). Nevertheless, we cannot rule out the 2016). possibility that B. flexus may behave better in other soils. We recommend that further field trials be conducted with these strains, both independently and as a bacterial mixture. References The increase in maize growth, enhancement of shoot P content and bioavailability of P in the substrates can be attrib- Bago B, Pfeffer PE, Shachar-Hill Y (2000) Carbon metabolism and trans- uted to the capacity of PSB for P solubilization, as well as port in arbuscular mycorrhizas. 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