This work aimed to assess the ability of plant growth-promoting Bacilli isolated from wheat rhizosphere and rock phosphate mine soils to convert inorganic phosphate (Pi) from Moroccan natural phosphate (NP) to soluble forms. The effect of these bacteria on wheat plants in order to increase their phosphorus (P) uptake in vitro was also investigated. Bacteria were isolated from wheat rhizosphere and natural rock phosphate soils and screened for their ability to solubilize Tri-Calcium Phosphate (TCP) and Natural Rock Phosphate (NP), to produce indole-3-acetic acid (IAA), siderophores and 1-aminocyclopropane-1-carboxylate (ACC) deaminase. Isolates were identified by 16S rRNA sequencing and tested for their capacity to increase wheat plants growth and their phosphorus uptake.Twenty-four strains belonging to Bacillus genus isolated from both biotopes were screened for their ability to solubilize Pi. The highest NP solubilization was showed by strains isolated from wheat rhizosphere. Solubilization of Pi was accompanied by organic acid production. Strains produce IAA, siderophore and ACC deaminase. Inoculation assays using efficient NP-solubilizing bacilli strains from both sources showed the ability of these isolates to increase wheat growth and the phosphorus uptake under in vitro conditions. Bacilli strains isolated from rhizosphere soil and natural rock phosphorus soil showed effective solubilization of Pi from rock phosphate. Phosphate solubilizing Bacilli were evaluated for their plant growth promotion under in vitro conditions. Results revealed the positive effect of all strains on biometric parameters and P content of wheat seedlings.
Modern Agriculture is facing many challenges where scientific communities are expected to play key roles. The world population is expected to reach 8000 million by 2020 and 9400 million in 2050 . The global food demand is increasing and there is a need to produce sufficient and healthy food. However, arable land degradation continues through a wide range of processes that make achieving sustainable food security challenging for several developing countries. Furthermore, water scarcity and climate change affect the global food production that is also unbalanced between regions and countries. In Morocco for example, cereal production represents about 15–20% of agricultural crop production . In Morocco, wheat (Triticum aestivum L.) is one of the major agricultural crops and with a production of 2.73 million tons in 2016 (www.fao.org/faostat) and 2.68 during 2018–2019 (https://www.onicl.org.ma). Modernization of Agriculture by using eco-friendly tools must be the driving force for innovation in agricultural systems. One of the biological tools that are considered as a boon for agriculture is the use of microbial inoculants with high plant growth promotion potential, particularly through solubilization of insoluble phosphate forms and to make phosphorus available to crops.
After nitrogen, phosphorus (P) is the second major key macronutrients in several biological processes for plant growth and development, including photosynthesis, energy transfer and nutrient uptake in plants.. However, extensive agriculture implies extensive use of phosphorus fertilizers, produced by chemical processing of high-grade rock phosphate ore (RPO) . Unfortunately, a considerable amount of the applied phosphorus based chemical fertilizers becomes usually unavailable to plants. In fact, plants for maximum productivity require approximately ~ 30 µmol L−1 of the applied phosphorus chemical fertilizers and the rest becomes unavailable as it is rapidly immobilized and precipitated with cations such as Ca2+, Fe3+and Al3+ . On another hand, the application of low-grade rock phosphate cannot be used directly in the soil, particularly in non-acidic soils such as moroccan soils. Hence, to improve the efficient use of Rock phosphate as alternative P source for important agronomic crops , Phosphate solubilizing microorganisms (PSM) could be used . Rhizosphere microorganisms or rhizobacteria are known for their positive effects on plant growth and are named plant growth promotion-rhizobacteria (PGPR) [7, 8]. PGPR are known to improve plant growth by phosphate solubilization, enhancing nutrient uptake by plants,and by phytohormones, siderophores and 1-aminocyclopropane-1_carboxylate deaminase (ACCD) production.
The purpose of this work was to evaluate plant growth traits of Bacillus spp. strains isolated and screened from two different habitats (wheat Rhizosphere soil and natural Moroccan Rock Phosphate (NP). Firstly, strains were characterized for their capacity to solubilize the inorganic phosphate forms: Tri-Calcium Phosphate (TCP) and NP. Secondly, their ability to produce indole-3-acetic acid (IAA) and ACC deaminase was determined. Finally, their effect on wheat plant growth, particularly efficient use of NP for P uptake under in vitro conditions was evaluated.
Material and Methods
Collection of Rhizosphere Soil and Rock Phosphate Samples
The rhizosphere soil samples of wheat were collected at different sites in North Morocco (Sidi Allal Tazi region, Kénitra Province). The plants were dug out, and soil adhering to the roots was collected aseptically in sterilized bags. Natural Moroccan Rock phosphate (NP) was sampled from extracted rock phosphate stockpiles located in the Ben Guerir mine of OCP, South Morocco. The samples were stored at 4 °C for further analysis.
Isolation of Phosphate Solubilizing Bacteria
One gram (1 g) of each of the two soil samples were transferred and homogenized in sterilized distilled water (9 mL). An enrichment step in (TSB) medium was necessary for rock phosphate samples before isolation. Soil suspensions were serially diluted (10–2–10–9) and aliquots were inoculated by spreading 0.1 mL on the modified Pikovskay’s (PVK) agar plates containing TCP as P source: Dextrose 10 g L−1, Ca3(PO4)2 (TCP) 5 g L−1, KCl 0.2 g L−1, NaCl 0.2 g L−1, MgSO4.7H2O 0.1 g L−1, MnSO4.H2O 0.002 g L−1, FeSO4.7H2O 0.002 g L−1, (NH4)2SO4 0.5 g L−1 . The plates were then incubated for 5 days at 30 °C. The colonies with clear halos indicating P solubilization were streaked on TSB and purified. Bacterial cells were gram stained and observed under the microscope for endo-spores. For long-term preservation, each isolate was stored at -80° in TSB containing 30% (v/v) glycerol.
Morphological, Biochemical and Molecular Characterization of Bacilli Strains
The isolated bacterial strains were identified on the basis of partial 16S ribosomal DNA (16S rDNA) sequences. The genomic DNA of bacterial strain was extracted by PureLink™ Genomic DNA Mini Kit (Invirogen, K182001). PCR reaction was performed using DreamTaq DNA Polymerase PCR Master Mix comprising 1 µg DNA, 0.4 mM dNTPs, 1 µM each primer, 4 mM MgCl2, (Invirogen, K1071) in a final reaction volume of 25 µL. Two universal primers, 27F 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R 5′- ACGGTTACCTTGTTACGACTT-3′  were used to amplify the 1500 bp 16S rDNA. The thermocycling conditions involved an initial denaturation at 95 °C (1–3 min), followed by 35 cycles of 95 °C for 30 s, 53 °C for 30 s and 72 °C for 1 min and a final extension at 72 °C for 15 min. PCR products were confirmed on 1% agarose gel and purified using PureLink Quick Gel Extraction Kit (Invitrogen, K220001). PCR products were sequenced in Secugen S.L. (https://www.secugen.es) and the obtained sequences were compared with those available in the EZTaxon server (https://www.eztaxon.org) and deposited in GenBank.The Phylogenic tree was constructed by neighbor-joining method using the MEGA 6.0 software.
Quantitative Assays for the Characterization of Plant Growth-Promoting Traits
Quantitative Determination of Phosphate Solubilization in Liquid Media
Phosphate solubilization ability of the isolates was performed in liquid medium with rock phosphate. The selected Bacilli isolates from rhizosphere soil and NP samples were further screened for their ability to solubilize natural rock phosphate as sole phosphate source in modified PVK medium. Quantitative analysis was carried out in Erlenmeyer flasks (250 ml) containing 50 ml of NP (5 g L−1)-PVK inoculated with each isolate at approximately 3.108 CFU mL−1 before incubation at 30 °C on a rotary shaker (150 rpm) for 5 days. Uninoculated broth served as control. The experiment was performed in three repetitions. After 5 days of incubation, the pH value of the medium was determined. The supernatant of each samples obtained by centrifugation at 1610 rcf for 15 min and inorganic phosphate content which carried out using automated Continuous Flow Analyzer (CFA)(Skalar, Netherlands) based on molybdenum blue method lies in the reaction between orthophosphate ions with ammonium heptamolybdate and potassium antimony(III) oxide tartrate forming the antimony-phospho-molybdate complex. This latter is intensely reduced to a blue color by ascorbic acid, then the standard curve of potassium dihydrogen phosphate (KH2PO4) was prepared in the range of 0.2; 0.4; 0.6; 0.8 and 1 mg P L−1.
UPLC Analysis of Organic Acids
The determination of organic acids produced by rhizosphere and rock phosphate soils strains were carried out by Ultra Performance Liquid Chromatography (UPLC) (Acquity, waters). 5 µL of filtered culture supernatants by 0.45 µm Millipore filter was injected into UPLC column C18 (4.6 × 250 mm, 5 µm). The mobile phase consisted of 0.1% phosphoric acid (H3PO4) solution, at a flow rate of 0.8 mL min−1. The column eluates were detected using a UV/Vis detector at 210 nm. Identification of each acid was performed by comparing retention time and peak areas with those of standards of organics acids including gluconic, citric, malic, lactic, propionic and succinic (Supelco, 47264).
Indole Acetic Acid (IAA) Production Assay
The production of IAA by rhizospheric and rock phosphate soil strains was determined using a standard method . The isolates were cultivated individually by adding 100 µL of overnight bacterial culture (107 CFU mL−1) in LB liquid medium containing 0.1 g L−1 of l-Tryptophan. After incubation at 30 °C in an orbital shaker (150 rpm for 3 days), each broth culture was centrifuged at 4000 rpm for 15 min and the supernatant was quantified spectrophotometrically at 535 nm using Salkowski reagent (0.5 M FeCl3:70% perchloric acid/water (2:49:49 ratio)). The concentration of IAA (μg mL−1) in the cultures was estimated by comparison with a standard curve (0; 5; 10; 20; 50 and 100 µg mL−1) of IAA (Fluka).
Quantitative Determination of ACC Deaminase Activity
ACC deaminase activity was determined calorimetrically following the standard method described by Penrose and Glick  by measuring the production of α-ketobutyrate and ammonia generated by enzymatic hydrolysis of ACC. Bacillus sp. isolates were cultivated overnight at 30 °C in nutrient broth (NB) under shaking at 150 rpm. The bacterial cells were collected by centrifugation at 6000×g for 10 min at 4 °C. Cell pellets were washed with 5 mL of DF salts minimal medium. After centrifugation for 10 min at 6000×g, the cells were suspended in 7.5 DF salts minimal medium containing a final concentration (3 mM) of ACC, as the source of nitrogen, and incubated at 30 °C for 24 h. After incubation, the supernatant was removed and cell pellets were subsequently washed with 5 ml of 0.1 M Tris–HCL (pH 7). The content was transferred in a 1.5 mL microcentrifuge tube, centrifuged for 5 min at 12000×g. The pellets were suspended and washed a second time in 600 μL of 0.1 M Tris–HCL (pH 8.5). Finally, 30 μL of toluene was added and the mixture was vortexed for 30 s. 200 μL of toluenized cells were mixed with 20 μl of 0.5 M ACC, vortexed and incubated for 15 min at 30 °C. Following the addition of 1 mL of 0.56 M HCL, the mixture was vortexed and centrifuged for 5 min at 12000×g. 1 mL of the supernatant was mixed with 800 μL of 0.56 M HCL. 300 μL of 2.4-dinitrophenylhydrazine (prepared in 2 M HCL) was added to the mixture and incubated for 30 min at 30 °C. Finally, 2 mL of 2 N NaOH was added to the reaction mixture and the absorbance was measured at 540 nm. After determining the amount of protein according to standards protocol , the concentration of α-ketobutyrate in each sample was determined by comparison with a standard curve. Results were expressed as micromoles of α-ketobutyrate per milligram of protein per hour (μmol L−1 α-KB mg−1 h−1).
Qualitative Assay for Siderophore Production
The siderophore production of rhizosphere and rock phosphate bacterial soils was detected using the universal method of . Briefly, the bacterial isolates were inoculated onto a blue agar plate containing the Chrome Azurol S (CAS). After incubation for 5–7 days at 30 °C, the CAS reaction was determined based orange halo around the colonies.
In vitro Assay for Plant Growth and Inoculation in Controlled Conditions
Wheat Seed Bacterization
To better study the effect of selected Bacilli isolates on wheat plant growth and P nutrition using NP as P source, four isolates from rhizosphere soil and two from natural phosphate with different phosphate solubilization activities were selected for this assay. Each strain was grown overnight in 50 mL of Nutrient Broth (NB) medium at 30 °C under shaking in an orbital incubator shaker at 150 rpm. Bacterization of wheat seeds was prepared as follow: Wheat (Triticum aestivum L. var. FARAJ) seeds were surface sterilized by chlorine gas (3.5% of NaClO containing 2.5% of HCL) for 15 min, rinsed in sterile distilled water and dried in sterile conditions. Carboxymethyl cellulose (CMC) 0.5% (w/v) was prepared and mixed with bacterial cells. The mixture was added to wheat seeds under shaking until a fine coating appeared on seeds. The control consisted of cell-free seeds coated with CMC. The coated seeds were assessed for the number of bacterial cells per seed and germinated on sterile paper at 25 °C for 2 days. Wheat plant growth experiments were further performed under in vitro conditions with natural phosphate (NP) as sole P source.
Wheat Plant Growth Under in vitro Conditions
In vitro growth experiment was conducted in the Murashige and Skoog (MS) medium . The experimental design consisted of eight replications in glass tubes containing 15 mL (0.13% agar w/v) of plant nutrition solution (phosphorus free). The dose of NP was 0.075 mg/tube/seedling. The experiment was designed to perform eight different treatments. Three control (without bacterial inoculation): MS P-free (control 1), MS + Natural phosphate (NP) (control 2), standard MS (control 3) and five treatments consisting of inoculated wheat plants and NP as P source: MS + NP + SM0307, MS + NP + SS0303, MS + NP + SS0306, MS + NP + RP10 and MS + NP + RP5. Thereafter the glass tubes were placed in phytotron at 26 °C, photoperiod of 16:8 h with an illumination intensity of 240 μmol m−2 s−1. After 7 days, the wheat plants were collected and the growth parameters such as plant length and root and shoot weight were measured. Approximately 0.4 g of plant samples were transferred to digestion tube and the digestion mixture (Sulfuric acid-Selenium) was added to plant samples. The digestion tubes were transferred to block digester preheated to 300 °C. The phosphorus uptake by plant was the calculation of P plant content using automated San + + Continuous Flow Analyzer (Skalar, Netherlands).
The statistical analysis and significant differences among means for bacterial PGP traits results were evaluated by ANOVA (P < 0.05) using Minitab version 16 statistical software and means were compared by Fisher’s test. In vitro assay data and correlations analysis between NP solubilization and organic acid peaks data were subjected to ANOVA using Fisher’s protected LSD and Multiple Correspondence Analysis (MCA) respectively by using SPSS version 22.
Isolation of Bacilli Strains
Bacillus spp. strains were isolated and selected from rhizosphere soil and rock phosphate mine soil by selective media. A total of 60 colonies were isolated from rhizospheric soil for which only14 isolates were selected. A number of 10 isolates from rock phosphate mine soil were selected. Table 1 showed the morphological characteristics of the selected bacteria.
Quantitative Assay of Phosphate Solubilization and Organic Acid Detection
The isolated strains were further screened to confirm their phosphate solubilizing efficiency by quantitative analysis in modified PVK liquid medium supplemented with different inorganic P sources. Among the 60 strains isolated from the rhizospheric soil, 14 isolates showed the highest amounts of phosphate solubilization (TCP) ranging from 12.73 ± 7.07 to 362.33 ± 2.89 mg L−1. For rock phosphate mine group, all the ten isolates showed TCP solubilization ranging from 39.55 ± 2.33 to 291.40 ± 8.91 mg L−1. When NP (Moroccan natural phosphate) was used as a sole P source, maximum solubilization was observed with the rhizosphere strains SM0307, SS0303, and SS0306 releasing 281.47, 253.67 and 254.20 mg L−1 soluble P in liquid medium, respectively (Table 2). Three phosphate mine isolates, on the other hand, showed a high level of phosphate solubilization: RP5 (41.4 mg L−1), RP6 (37.53 mg L−1) and RP10 (32.83 mg L−1) (Table 3). The phosphate solubilization in liquid medium was accompanied by decreasing in pH. No significant negative correlation between TCP solubilization and pH for rhizosphere strains (r = − 0.38; P > 0.01) and for rock phosphate mine strains (r = − 0.48;P > 0.01) was observed. However, Significant (P < 0.01) and negative correlation was observed between the NP solubilization and pH measurement (r = − 0.78) and (r = − 0.92) for rhizosphere and rock phosphate mine isolates, respectively.
The analysis of organic acids in the supernatant was performed in bacterial culture filtrate using UPLC after 5 days of incubation for isolates showing pH decrease in NP solubilization assay (Fig. 1) presented chromatograms of standard organic acids produced by three strains isolates from each soil sample. Retention times for all isolates were compared to those of standards. The analysis showed that malic acid peaks were detected in nine isolates. The rest of the retention times peaks might represent unknown organic acids.
Quantitative Assay of Indole-3-Acetic Acid (IAA), ACC Deaminase and Siderophore Production by Bacilli Isolates
Indole-3-acetic acid production was quantified in broth culture using Salkowski reagent. Both groups of bacterial isolates produced indole-3-acetic acid (IAA) in the LB medium supplemented with tryptophan. Regarding the rhizosphere strains, 12 of the 14 isolates had the ability to produce IAA in LB medium with concentrations ranged from 2.50 ± 0.83 to 10.99 ± 0.24 µg mL−1 (Table 2). Similarly, 10 bacterial isolates from rock phosphate mines were able to produce IAA, with concentrations varied from 3.24 ± 1.56 to 12.66 µg mL−1. The maximum IAA concentrations were observed in three isolates, RPS8 (12.66 ± 1.76 μg mL−1), RPS3 (12.13 ± 2.33 μg mL−1) and RPS10 (11.08 ± 0.13 μg mL−1) (Table 3). The ACC deaminase enzyme activity was determined based on the quantitative assay of α-ketobutyrate for different Bacillus spp. isolates. All isolates showed their ACC deaminase capacity with varied concentrations ranging from 0.207 ± 0.14 to 3.068 ± 0.54 μmol mg−1 protein h−1 for rhizosphere isolates and from 0.339 ± 0.00 to 3.223 ± 0.00 μmol mg−1 protein h−1 for phosphate mine isolates. The two groups of Bacilli isolates were studied qualitatively for siderophore production. Out of the 24 isolates tested in CAS-agar plate, 22 strains were siderophore positive (Tables 2 and 3).
Molecular and Biochemical Identification of Bacilli
Bacilli isolates were identified based on the analysis of the PCR-amplified 16S rDNA sequences (Table 4). The obtained sequences were deposited in Genbank and compared to sequences available in databases using Ez-Taxon database (Table 4). 16S rDNA phylogenetic trees were generated by neighbor-joining using Mega.v6, and showed that the studied strains belong to the genus Bacillus spp (Supplementary material, Fig. 1a and b).
Plant Growth Promoting Assay Under In vitro Conditions
Wheat plants were harvested after 7 days and analyzed for different growth parameters, root and shoot length and weight and phosphorus content. Wheat seeds coated with different phosphate solubilizing Bacillus spp isolated from rhizosphere and rock phosphate mine showed a significant increase in plant growth parameters (Fig. 2). Results showed a statistically significant increase (P < 0.05) of wheat root and shoot length of bacilli inoculated plants in comparison to control plants (Table 5). Higher and vigorous seedlings were obtained with inoculation with the selected Bacilli and values ranged between 6.33 ± 2.48 cm and 7.33 ± 3.53 cm for toot length and between14.05 ± 3.04 cm and 16 ± 3.22 cm for shoot length (Table 5, Fig. 2). The control consisting of uninoculated plants, cultivated in MS medium and MS P-free or supplemented with NP as P source showed lower values (3.96 ± 2.57 cm and 12.91 ± 1.98 cm for root and shoot length, respectively). Root and shoot biomasses were also significantly improved in inoculated plants compared to the control treatments (Table 5). Root and shoot fresh weights of plants inoculated with the isolates showed enhancement by two fold compared with plants cultivated in MS without P or supplemented with NP (Table 5).
Interestingly, P contents increased in wheat seedlings treated with Bacilli in the presence of NP over control (Fig. 3). Among the bacterial treatment, plant phosphorus content increased more significantly in plants inoculated with rhizospheric Bacilli (SS0303, SS0303 and SM0307) than in plants inoculated by rock phosphate mine strains (RP10 and RP5). Moreover, rhizospheric strains increased the P content in wheat plants in comparison to plants inoculated with rock phosphate strains. The bacilli seem to contribute to P nutrition of wheat plants and increase the P efficiency of the natural Moroccan rock phosphate.
Inorganic phosphate solubilizing bacteria have been characterized in many reports. In this work, bacilli were screened from two different environments, wheat rhizosphere and Moroccan phosphate rock, for their inorganic phosphate solubilizing ability and deeply investigated for their contribution to plant P uptake. The role of Bacillus from Moroccan phosphate rock is still poorly investigated. Bacillus sp. is an ubiquitous genus in the Bacillaceae family with a high capacity to survive in extreme and diverse habitats ranging from water and soil to extreme environments . These gram-positive spore-forming bacteria can colonize plant roots and enhance nutrients availability and hence their application inagrobiotechnology, could be relevant for the future of sustainable agriculture [18,19,20].
Members of Bacillus also hold other plant growth promoting traits such as siderophore and phytohormones production and induction of systematic resistance in plants leading to defence responses against phytopathogens [18, 19, 21]. Bacteria belonging to Bacillus megaterium var phosphaticum were reported to be able to increase phosphate solubilization and mineral uptake leading to plant growth enhancement of pepper and cucumber . Brevibacillus Brevis could improve in vitro spore germination and growth of the arbuscular mycorrhizal fungus Glomus mosseae by increasing the presymbiotic growth . This positive association between the bacterium and the fungus could be associated with an increase in plant nutrition.
In the present study two groups of Bacillus spp. strains isolated from rhizosphere and rock phosphate mines soils were used for phosphate solubilization assay. The driven force for the choice of these habitats was the need to enhance the agronomic efficiency of Moroccan rock phosphate by selecting PSM that can use this insoluble P source for plant fertilization. Firstly, tri-calcium phosphate (TCP) was used as sole phosphorus source (initial screening of phosphate solubilizing bacteria)  on pikovskaya agar medium followed by checking for their ability to show halos around the PSM colonies corresponding to P solubilization. In addition, all the bacterial isolates from both habitats were assessed for their P solubilization abilities in pikovskaya liquid medium. A number of 60 strains were obtained from rhizosphere soil and 10 strains from rock phosphate mines. However, only 14 isolates from the rhizospheric soil; SS0303, SM0112, SS0101, SS0304, SM0307, SS03006 and SS0307 which showed maximum solubilization of tri-calcium phosphate (TCP)(P < 0.05) ranged from 111.17 ± 6.62 to 362.33 ± 2.89 mg L−1 were kept. Regarding the TCP solubilization by the rock phosphate mine strains, most of isolates showed maximum solubilization (P < 0.05) which ranged between 102.05 ± 1.63 and 281.50 ± 5.94 mg L−1. The capacity to easily solubilize TCP by bacterial strains in liquid medium was reported by numerous studies [18, 20, 25]. However, the reliability of this screening assay is very often criticized. Indeed, some research works reported that many bacteria could solubilize important quantities of P in broth despite they not show halo zone in PVK plate . Besides, various works have demonstrated that phosphate solubilizing capacity of some bacteria and fungi differ according to P source and indicated that TCP could be inappropriate as a universal screening factor for isolating and testing phosphate solubilizing bacteria . In the present study, Moroccan natural phosphate (NP) was also used as a phosphate source and significant differences (P < 0.05) were observed for NP solubilization values for rhizosphere soil isolates. The strains SM0307, SS0303 and SS0306 were the most efficient, and released the highest amount of phosphorus in the medium of 281.47 ± 15.82 mg L−1, 253.67 ± 56.75 mg L−1 and 254.20 ± 23.90 mg L−1, respectively. Another study by Chang et al.  reported that Bacillus smithi F18 was able to release P amount of 544.2 ± 30.2 mg L−1 from inorganic phosphate TCP and 451 ± 24.9 mg L−1 from natural phosphate. Compared to rhizopheric soil strains, all rock phosphate mine isolates solubilized natural phosphate (NP) in liquid medium to a lesser extent than TCP (amounts ranging from 5.56 ± 1.33 to 41.40 ± 1.95 mg L−1). The high abilities to solubilize TCP in comparison with NP could be due to their structural differences. Indeed, TCP has a simple structure, compared to natural phosphate which is less easily solubilized by phosphate solubilizing bacteria (PSB) . Similarly, the difference observed for rhizosphere soil isolates and rock phosphate soil isolates in their differential solubilizing abilities is probably due to their different isolation origin . In the present study, the results of soluble P content in natural phosphate NP(5 g L−1) -PVK medium under in vitro condition (Figs. 4 and 5) demonstrate that rhizospheric soil strains possess a high NP solubilization capacity, even compared to rock phosphate mine strains and to other phosphate solubilizing Bacillus sp. soil bacteria . Compared to the non-rhizosphere soil, the rhizosphere zone is an important source of organic root-born carbon, which is needed for soil bacterial metabolism. On another hand, this organic carbon is considered as one of the essential elements responsible for the heterogeneous microbial communities in the rhizosphere. In other study, Reyes et al.  studied the hydroxyapatite solubilization by phosphate solubilizing microorganisms isolated from rhizospheric soils, bulk soils and rock phosphate mines. The authors succeded to isolate a large number of strains from rhizospheric soil showing higher hydroxyapatite dissolution compared to bulk soil isolates.
During natural phosphate solubilization assay, a significant decline in pH was observed with rhizospheric strains SS0303 (3.92 ± 0.02) and SM0307 (3.93 ± 0.017), compared to the three rock phosphate mine strains RPS10, RPS5 and RPS6 which showed a final pH of 4.75 ± 0.02, 4.80 ± 0.06 and 4.91 ± 0.05, respectively. It is well known that acidification of the medium is one of the important mechanisms associated with the release of P from insoluble complexes in liquid medium [27, 30]. The production of low molecular weight organic acids during the release of soluble natural phosphate (NP), including gluconic acid, 2-ketogluconic acid, malic acid, citric acid, propionic acid, and succinic acid was demonstrated as directly associated with pH decrease in the medium . Among all of the organic acids generated by different phosphate solubilizing bacteria, gluconic acid is the most frequent acid produced during phosphate solubilization by bacteria . In the present study, we showed a negative correlation between pH and phosphate solubilization, suggesting the role of organic acid in this solubilization process. The analysis of organic acids in supernatants of some cultures using UPLC allowed us to identify the production of malic acid by nine isolates. However, the correlation matrix (Table 6) between P solubilization and retention times revealed a positive correlation with two unknown organic acids detected in 1.13 min and 1.23 min, respectively. Similar interesting observations were showed by another study by Chen et al. . The absence of correlation of different known organic acids has been identified in others study (e.g., gluconic acid) . These results suggest that other organic acids than those used in the present assay can facilitate phosphate solubilization from natural phosphate. Further studies are needed to identify these compounds.
Plant growth-promoting traits of PGPR can be achieved by other activities other than phosphate solubilization. Therefore, rhizospheric and rock phosphate mine bacilli were evaluated for phytohormones production. Phytohormones are considered fundamental compounds for plant growth and development such as cell division and root elongation. The most common phytohormones produced by PGPR is Indol-3acetic acid (IAA) which could be produced in vitro by supplementing the LB medium with its precursor, L-tryptophan . In this study, results of the quantitative assay of IAA production revealed that most isolates produced IAA, except two rhizospheric soil isolates that were not able to produce this phytohormone. The production amounts of IAA ranged from 2.50 ± 0.83 μg mL−1 to 12.66 ± 1.76 μg mL−1. Most Bacillus spp. strains were reported as able to produce IAA in the presence of l-Tryptophan [37, 38]. in addition, many reports indicated a variability in IAA production under growth conditions and also a variability between genera and strains of the same species [39, 40]. Besides phosphate solubilization and indole-acetic acid production, another PGPR trait that can help plants under environmental stresses is the Production of 1-aminocylopropane-1-carboxylate (ACC) deaminase. This enzyme induced enzymatic hydrolysis of ACC, a precursor for ethylene production by plants, to α-ketobutyrate and ammonia, resulting in the decrease of ethylene levels in plants and consequently decreasing the stress harmful effects . In this study, results obtained for all studied Bacillus spp. strains showed the ability to use ACC as the sole nitrogen source at 3 mM to produce ACC deaminase. Similarly, higher amounts of α-ketobutyrate, 1.85 μM, and 0.73 μM for Bacillus sp strains have been reported by Baig et al.  and Lim et al. , respectively. However, higher ACC deaminase activity in Bacillus spp. strains was also reported; 54.14 μmol of α-KB.mg−1 Pr h−1  and 267.50 nmol of α-KB.mg−1 Pr h−1 . Noreen et al. , reported an enzymatic activity of up to 355 nmol of α-KB/mgPr/h Pseudomonas sp.. However, it was demonstrated that levels of ACC deaminase activity ≥ 20 nmol α-KB.mg−1 Pr h−1 are sufficient to enhance high levels of ethylene in plant under stress and to act as a PGPR .
Under in vitro conditions, wheat plants were grown on a semi-liquid medium enabling supply of nutrients directly to the roots. In this study, Murashige and Skoog medium has been used along with Moroccan natural phosphate (NP) as a sole source of P for plants after inoculation with selected Bacilli. Phosphate solubilizing bacteria (PSB) with their PGP traits can increase the uptake of a nutrient elements like phosphorus . In the present study, significant increases were observed in biometric parameters (shoot and root length and weight) of the wheat plant due to coating seeds with selected Bacilli compared to control plants (Table 5). Treatments that combined rhizospheric strains (MS + NP + SM0307) or rock phosphate mine strains (MS + NP + RP10) (MS + NP + RP5) with natural phosphate were led to a significant increase in shoot biomass compared to control treatments, particularly those consisting of MS P-free and MS supplemented with NP (MS + NP). Han et al. showed that PSB Bacillus sp. increased roots and shoots biomass with natural phosphate compared to control. It is likely that bacterial strains promote shoot and root growth. Plant growth-promoting effects depend on direct effects towards plant mainly by facilitating nutrients uptake particularly phosphorus nutrients. Regarding P content in the plant, the bioavailability of P in growth medium showed a high correlation with P uptake in the wheat plant. In our study, all of bacterial coated seeds treatment along with natural phosphate (NP) showed a significant increase in amounts of total P content in the wheat plant and were particularly more pronounced in seedlings inocumated with rhizospheric strains (Figs. 2, 3). Similar results were reported by Sarker et al.  in wheat seedling inoculated with Pseudomonas and cultivated in the presence of TCP and by Swaranalakshmi et al. and Hamdali et al. in wheat plants inoculated with phosphate solubilizing bacteria (PSB) and supplemented with natural phosphate (NP) showing a significant increase of P content in comparison with control plants. Results of wheat plants grown under in vitro conditions were in accordance with the quantitative assay of biosolubility of P from rock phosphate demonstrating a significant increase in phosphate solubility by rhizospheric Bacilli when compared to rock phosphate mine strains. These results suggested that rhizospheric Bacilli can affect plant growth by a different mechanism including phosphate bioavailability and can significantly contribute to increasing the agronomic efficiency of NP.
In summary, fourteen bacterial strains from wheat rhizospheric soil and 10 strains from rock phosphate soil were selected in the first step by their phosphate solubilization abilities in addition to other PGP traits: indole-3-acetic acid (IAA), siderophores and ACC deaminase production. Most isolates produce siderophores. These compounds could be involved in Fe nutrition but also they were reported in other species as involved in P solubilization. These isolates were identified by 16S rDNA gene sequencing as belonging to the genus Bacillus spp. Thereafter, we screened efficient phosphate solubilizing bacilli: 3 rhizospheric strains and 3 rock phosphate mine strains according to their high levels of P solubilization using NP in a liquid medium. 55 different unknown compounds were detected in the supernatant of culture medium by UPLC analysis to assess the role of medium acidification on the P solubilization activities of these Bacilli. The focus was kept on natural phosphate solubilizing bacilli that were evaluated for their plant growth promotion under in vitro condition. Results revealed the positive effect of all strains on biometric parameters of wheat plant root and shoot. Moreover, the inoculation of wheat plants with rhizospheric Bacilli in combination with natural phosphate (NP) showed “good” results in the P content of plants. We demonstrated that these Bacilli can enhance the efficient use of NP as a source of P-fertilizer. Further studies could be performed to assess the role of these bacilli under greenhouse and field conditions, and evaluate more physiological plant traits such as chlorophyll, proteins, and enzymatic activities when wheat plants are put to grow in the presence of natural phosphate. On other hands, we noticed a more solubilizing capacity of rhizosphere soil adapted Bacilli. This encourages us to screen for strains from the rhizospheric soils of native plants in the phosphate mines. This could help in the understanding of the differences obtained between the two groups of Bacilli in this study. Our results are encouraging and suggest the potential of the isolates for sustainable wheat productivity.
Natural rock phosphate
- ACC deaminase:
1-Aminocyclopropane-1-carboxylate (ACC) deaminase
Rock Phosphate Ore
Ultra performance liquid chromatography
Phosphate solubilizing microorganisms
Plant growth promotion-rhizobacteria
Tryptone soy broth
Murashige and skoog
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The Authors would like to acknowledge the support through the R&D Initiative – Appel à projets autour des phosphates APPHOS – sponsored by OCP (OCP Foundation, R&D OCP, Mohammed VI Polytechnic University, National Center of Scientific and technical Research CNRST, Ministry of Higher Education, Scientific Research and Professional Training of Morocco MESRSFC) under the project entitled * Bioformulation de Consortium de Microorganismes solubilisateurs du phosphate: effets bénéfiques sur la croissance et la protection des plantes *, project ID * BIO-BIZ-01/2017*”
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Azaroual, S.E., Hazzoumi, Z., Mernissi, N.E. et al. Role of Inorganic Phosphate Solubilizing Bacilli Isolated from Moroccan Phosphate Rock Mine and Rhizosphere Soils in Wheat (Triticum aestivum L) Phosphorus Uptake. Curr Microbiol 77, 2391–2404 (2020). https://doi.org/10.1007/s00284-020-02046-8