Acta agriculturae Slovenica, 117/3, 1–14, Ljubljana 2021 doi:10.14720/aas.2021.117.3.1874 Original research article / izvirni znanstveni članek The possible use of scarce soluble materials as a source of phosphorus in Vicia faba L. grown in calcareous soils Abdelmonem Mohamed ELGALA 1 and Shaimaa Hassan ABD-ELRAHMAN 1, 2 Received September 13, 2020; accepted August 06, 2021. Delo je prispelo 13. septembra 2020, sprejeto 6. avgusta 2021 1 Soil Science Department, Faculty of Agriculture 11241, Ain Shams University, Egypt 2 Corresponding author, e-mail: Shaimaa_Hassan@agr.asu.edu.eg The possible use of scarce soluble materials as a source of phosphorus in Vicia faba L. grown in calcareous soils Abstract: Phosphorus (P) is affected by many factors that minimize its solubility especially in calcareous soils. The aim of this work was to conduct laboratory and greenhouse experiments to study the effect of using P solubilizing sub- stances, i.e., compost, humic acid (HA), citric acid and eth- ylene di-amine tetra acetic acid (EDTA), and rhizobacteria, Bacillus megaterium var. phosphaticum on solubilizing P from different sources, ordinary superphosphate (OSP), rock phos- phate (RP) and basic slag (BS). The effect of these treatments on the P- availability in El-Nubaria calcareous soil and P- up- take by faba bean (Vicia faba ‘Giza 843’) were studied. Ob- tained results showed that the solubility of P sources differs in their ability to release soluble P in the following order: OSP > RP > BS. The following descending order was appeared of available P in soil with addition of solubilizing agents: citric acid > EDTA > HA > compost for these sources of P , for both experiments. Regarding the interaction between solubilizing agents, the treatments of HA combined with EDTA or citric acid were superior in giving high concentrations in soil, and vigor plant growth. In addition, the solubility of P increased by about 5-6 times for all sources in the presence of P- dis- solving bacteria. It seemed that the presence of appreciable amounts of Mg, S, Fe, Mn, B and other elements in BS played a role in enhancing plant growth and increasing yield, espe- cially in the presence of added bacteria. BS could be used in calcareous soils and for soils characterized by low nutrient supply as sandy. Key words: phosphorus sources; basic slag; organic sub- stances; chelating substances; P availability; P dissolving bac- teria; calcareous soils; Vicia faba Možnost rabe slabo topnih snovi kot vir fosforja pri gojenju boba (Vicia faba L.) na apnenčastih tleh Izvleček: Na topnost fosforja (P) vplivajo številni de- javniki, še posebej v apnenčastih tleh. Namen te raziskave je bil izvesti poskus v laboratoriju in rastlinjaku za preučevanje učinkov fosfor sproščajočih snovi kot so kompost, humin- ska kislina (HA), citronska kislina, etilen diamin tetra oce- tna kislina (EDTA) in rizobakterij (Bacillus megaterium var. phosphaticum) na topnost fosforja iz različnih virov kot so navaden superfosfat (OSP), fosfat v kamnini (RP) in toma- ževa žlindra (BS). Preučevani so bili učinki teh obravnavanj na dostopnost fosforja v apnenčastih tleh v El-Nubaria, Egipt in privzem fosforja v bob (Vicia faba ‘Giza 843’). Rezultati so pokazali, da se topnost fosforja iz različnih virov razlikuje gle- de na njegovo sposobnost sproščanja v naslednjem vrstnem redu: OSP > RP > BS. Po dodatku agensov za topnost se je v tleh pojavil naslednji padajoči redosled razpoložljivega P: citronska kislina > EDTA > HA > kompost, za vse vire fosforja v obeh poskusih. Glede na interakcije med agensi za topljenje se je obravnavanje HA v kombinaciji z EDTA ali citronsko ki- slino izkazalo kot najboljše, z največjo koncentracijo topnega P v tleh in najboljšo rastjo rastlin. Dodatno se je vsebnost P povečala za okrog 5-6 krat pri vseh virih P v prisotnosti fos- for sproščajočih bakterij. Zdi se, da je je prisotnost precejšnih količin Mg, S, Fe, Mn, B in drugih elementov v tomaževi žlin- dri vplivala na pospešeno rast rastlin in povečanje pridelka, še posebej ob dodatku bakterij. Tomaževa žlindra bi se torej lahko uporabljala na apnečastih tleh in v peščenih tleh, ki jih označuje majhna vsebnost hranil. Ključne besede: viri fosforja; tomaževa žlindra; organ- ske snovi; helatirajoče snovi, razpoložljivost P; P raztapljajoče bakterije; apnenčasta tla; Vicia faba Acta agriculturae Slovenica, 117/3 – 2021 2 A. M. ELGALA and S. H. ABD-ELRAHMAN 1 INTRODUCTION In Egypt, phosphorus (P) is the second major fer- tilizer comes after nitrogen and it is added to the soil mainly as an ordinary superphosphate (OSP). Phos- phorus is an insoluble element in alkaline soil especially in soils containing high calcium carbonate, e.g., calcare- ous soils, which causes rapid precipitation to insoluble phosphate forms (Elgala & Amberger, 2017). The defini- tion of calcareous soils, as reported by Hopkins & Ells- worth (2005), that are having significant quantities of calcium or magnesium carbonate (2-12 % depending on their particle size). These salts dissolve in neutral to acid soil pH (7-6.5), but not readily dissolve in alka- line soil (at about pH ≥ 8) and, instead, serves as a sink for surface adsorbed calcium phosphate precipitation. In other words, calcareous soils with high pH resulting from high content of salts or Na + and OH - ions, made P is a limiting factor, causing nutritional stress conditions. Many factors affect the solubility of P in soil and its availability to growing plants, particularly under P-stressed conditions: with using rock phosphate or other untraditional components as a source of P; such as acidifying the root medium (Houassine, 2020) and adding organic acids, amino acids and other chelating substances (Grover, 2003; Taskin et al., 2019; Elhag et al., 2019). Accordingly, getting benefit of factors that help in increasing solubility and availability of P from insoluble sources may encourage the use of rock phos- phate (RP) or recycling untraditional sources i.e., basic slag (BS), even under alkaline conditions, with preserv- ing the environment from contamination. Basic slag or steel slag, as common, contains calcium oxide (CaO, 40- 50 %) and silica (SiO 2 , 10-28 %). Also, it includes alumi- na (Al 2 O 3 , 1-3.5 %) and magnesium oxide (MgO, 2.5-10 %), as well as iron oxide (FeO, 14-22 %) and manganese oxide (MnO, 1.5-6 %), total Fe (17-27 %), and appreci- able amounts of P, K, S, and micronutrients (Tsakiridis et al., 2008; Yildirim & Prezzi, 2011; Bing et al., 2019). BS could be used in agricultural fertilizers, and envi- ronmental protection (Bing et al., 2019). Negim et al. (2010) found that the BS additions increased soil pH and conductivity, while immobilized Cu, Zn, Cr and Cd in the studied contaminated acid soil, which reflected on Phaseolus vulgaris L. growth. Also, Ning et al. (2016) reported that BS was an effective amendment for soil acidity adjustment, plant Si nutrition and stabilization of Cd in acidic soils. Humic acid (HA) is a common fertilizer con- taining most elements that improve soil fertility and increase nutrients availability, thus enhances plant growth, and yield as well as decreases the harmful effect of stresses (Doran et al., 2003). The effect of HA on the availability of P and micronutrients in calcareous soils have been given especial attention because of observed increases in uptake rates of these nutrients following application of HA (Satisha & Devarajan, 2005; Elhag et al., 2019). Also, compost is seen to be beneficial in improving soil fertility and crop productivity (Adugna, 2016), remediating polluted environment, recycling agricultural wastes (Taiwo, 2011), reducing the phyto- toxicity of heavy metals (Huang et al., 2016), increasing water use efficiency (Adugna, 2016), and microbial ac- tivity (Huang et al., 2016; Lee et al., 2019). In addition, organic chelating agents such as EDTA and citric acid, significantly enhance element solubility and uptake by plants (Afshan et al., 2015), and are commonly used as they are more effective in chelating elements and in- creasing their concentrations in the upper plant organs (Kanwal et al., 2014). Bio-fertilizers are playing a vital role in sustain- able agricultural management to reduce environmental contamination (Bulut, 2013). Bacillus megaterium var. phosphaticum, which is considered a rhizobacteria, can exert a positive effect on plant growth through solubi- lizing inorganic phosphate and mineralizing organic phosphate, helping P to be readily available to plants with time (Abd-Elrahman, 2016; Saxena et al., 2020). Due to the P solubilization capacity, B. megaterium var. phosphaticum could be used along with RP or any other natural source to raise their efficiency in the soil. These cells can produce amino acids, vitamins, indole acetic acid (IAA), gibberellic acids, antibiotics, siderophore, as well as organic and inorganic acids that mobilize P and other nutrients and encourage the plant growth (Cak- makci et al., 1999; Amalraj et al., 2012). In addition, for the mineralization of organic P compounds, it could be due to the release of phosphatase enzymes (Illmer et al., 1995; Płaza et al., 2021). So, the aim of this work was to conduct laboratory and greenhouse experiments to study the effect of using P solubilizing substances and rhizobacteria to solubilize P from different sources. The effect of these treatments on the P- availability in calcareous soils and P- uptake by faba bean plants (Vicia faba ‘Giza 843’) were also studied. 2 MATERIALS AND METHODS The current study involves two trial types: 2.1 INCUBATION EXPERIMENT To assess P content in a pure media (any salts, Acta agriculturae Slovenica, 117/3 – 2021 3 The possible use of scarce soluble materials as a source of phosphorus in Vicia faba L. grown in calcareous soils CaCO 3 and P were removed) treated by several scarce soluble materials, 200 g of acid (HCl 10 -4 M) washed quartz sand were placed in a plastic bowl, kept at the laboratory conditions (24 ± 2.5 o C). Thirty combinations generated from application of fifteen treatments either without adding bacteria or in the presence of dissolving bacteria (Bacillus megaterium var. phosphaticum) as fol- lows were tested with three replications: 1- Ordinary Superphosphate (OSP) 2- OSP + Compost 1 % 3- OSP + Humic acid 1 % 4- OSP + Citric acid 1 % 5- OSP + EDTA 1 % 6- Rock Phosphate (RP) 7- RP + Compost 1 % 8- RP + Humic acid 1 % 9- RP + Citric acid 1 % 10- RP + EDTA 1 % 11- Basic Slag (BS) 12- BS + Compost 1 % 13- BS + Humic acid 1 % 14- BS + Citric acid 1 % 15- BS + EDTA 1 % Extractable P concentration in each treatment and total element concentrations in basic slag were mea- sured before adding to the soil. The P sources, i.e., ordi- nary superphosphate (OSP), rock phosphate (RP) and basic slag (BS), were added at a rate of 4.0 g kg -1 sand (equal to 9.6 t ha -1 ). To meet the proper requirements as recommended by the Egyptian Ministry of Agriculture for faba bean cultivation in newly reclaimed soils (55.8 kg P 2 O 5 ha -1 in the form of ordinary superphosphate). Each of rock phosphate granules fertilizer (obtained from Abou Zaabal Company) and basic slag (obtained from Iron and Steel Company in Helwan) were ground to pass through a 2.0 mm sieve. According to the treat- ment, 20 ml bowl -1 of Bacillus megaterium var. phosphat- icum bacterial suspension, 1 x 10 9 cells ml -1 , (supplied by the Department of Microbiology, Faculty of Agricul- ture, Ain Shams University) were added. Tap water was added to keep the moisture of the medium at the field capacity till the end of the incubation period. Sand samples (20 g bowl -1 ) were taken 3 times; after 2, 4 and 8 weeks. The collected samples were air dried, crushed, sieved to pass through a 2.0 mm sieve, and prepared to determine available P spectrophoto- metrically using Olsen extract (0.5 M NaHCO 3 at pH 8.5) according to the method described by Watanabe & Olsen (1965). 2.2 POT EXPERIMENT A pot experiment was carried out in autumn sea- son of 2019 at the greenhouse of Soil Science Depart- ment, Faculty of Agriculture, Ain Shams University, Qalubia governorate, Egypt. The experiment was kept in air temperature (21.9 ± 3.8 °C). Representative soil samples were collected from the surface layers (0-20 cm) of a calcareous soil, Typic Torripsamments (accord- ing to Soil Survey Staff, 2010), sandy loam soil from El- Nubaria district (30º39ʹ55ʺ N and 30º41ʹ49ʺ E), Beheira governorate, Egypt. The polythene lined pots (18 cm in diameter and 15 cm in height) were packed uniformly with 3.0 kg of the investigated soil which was already air dried and ground to pass through a 2.0 mm sieve. Some initial physical and chemical properties of the studied soil were tested before plant cultivation according to the standard methods outlined by Page et al. (1982) and Klute (1986). The abovementioned 15 treatments plus 4 solubilizing agents’ treatments (compost, humic acid, citric acid and EDTA) and their 6 combinations (com- post+ humic acid, citric acid+ EDTA, compost+ citric acid, compost+ EDTA, humic acid+ citric acid and hu- mic acid+ EDTA) were added to the pots and mixed well with the soil during packing, with the same doses. Two control (check) treatments were also applied (one for soil free of P sources and without adding bacteria, and the other for soil free of P sources in the presence of dissolving bacteria). Tap water was used to keep the moisture of the soil before and after plant cultivation at the field capacity till the end of the experimental work. After one week from adding the treatments, pots were cultivated with faba bean seeds (Vicia faba ‘Giza 843’ , 5 seeds pot -1 ) on 16 th of October 2019. At the same time, according to the treatment, 20 ml pot -1 of Bacil- lus megaterium var. phosphaticum bacterial suspension (1 x 10 9 cells ml -1 ) were added. After seeds germination, plants were thinned to one plant pot -1 . Nitrogen fertiliz- er in the form of (NH 4 ) 2 SO 4 and potassium in the form of K 2 SO 4 were applied, at a rate of 1.0 g kg -1 soil (equal to 2.4 t ha -1 ) for each, in two batches the first one at the vegetative growth stage, 60 days after sowing (DAS), and the other one at the flowering stage (90 DAS). 2.3 MEASUREMENTS 2.3.1 Soil P content Soil in the investigated pots was sampled 4 times: (i) after seeds germination (14 DAS), (ii) at the vegeta- tive growth stage (60 DAS), (iii) at the flowering stage (90 DAS), and (iv) at plant harvest (145 DAS). The col- Acta agriculturae Slovenica, 117/3 – 2021 4 A. M. ELGALA and S. H. ABD-ELRAHMAN lected samples were air dried, crushed, sieved through a 2.0 mm sieve, and prepared to determine available P spectrophotometrically using Olsen extract, as de- scribed by Watanabe & Olsen (1965). 2.3.2 Crop traits Plants were harvested on the second week of March 2020, to assess plant height, plant fresh and dry mass, as well as number of pods plant -1 , fresh mass of pods and seeds plant -1 . Also, samples of plant leaves were oven dried at 70 ºC for 48 h and digested by H 2 SO 4 / H 2 O 2 mixture according to the method described by Chapman & Pratt (1961). Total nitrogen in leaves was determined using Kjeldahl method according to the procedure described by Chapman & Pratt (1961), total phosphorus was determined using Spectrophotometer according to Watanabe & Olsen (1965) and total potas- sium in plant leaves was determined using Flame pho- tometer as described by Chapman & Pratt (1961). 2.4 EXPERIMENTAL DESIGN AND STATISTICAL ANALYSIS The two experiments (incubation and pot experi- ments) were designed in a completely randomized de- sign and each treatment was replicated three times. The obtained data were then statistically analyzed using SAS software package (SAS, 2000). V alues expressed as mean and were compared for each other using Duncan’s mul- tiple range test (at p ≤ 0.05 considered significant) ± standard error of the mean (SEM, n = 3). 3 RESULTS AND DISCUSSION 3.1 INITIAL CHARACTERISTICS OF SOIL AND TREATMENTS Extractable P concentration in each treatment be- fore adding to the investigated soil are shown in Table 1a. The treatment of OSP is rich with P, followed by humic acid, RP, compost and BS respectively. Regard- ing the mixtures between treatments (Table 1a), the treatment of citric acid+ OSP gave high concentration of soluble P, followed by EDTA+ OSP, citric acid+ RP, EDTA+ RP, EDTA+ BS and citric acid+ BS respec- tively. Total element concentrations in basic slag were measured before adding to the soil (Table 1b). It seems good that finding appreciable amounts of P, Mg, S, Fe, Mn, B and other elements in BS. Some initial physical (soil texture, field capacity, wilting point, and satura- tion percent) and chemical (CaCO 3 fractions content, organic matter content, soil cation exchange capacity, pH, electrical conductivity of salts, soluble ions concen- tration, total and available concentration of macronu- trients NPK) properties of the studied soil before plant cultivation are presented in Table 2. The studied soil is calcareous sandy loam with no saline hazards and low macronutrients concentration. 3.2 INCUBATION EXPERIMENT Data in Table 3 shows the availability of P con- centrations in acid washed sand with time; after apply- ing different P- sources and solubilizing agents, with or without adding P dissolving bacteria. As P fertiliz- ers (OSP, RP and BS) which vary in their P contents were added to washed sand at equal rates (4 g kg -1 ), the extractable amounts in washed sand were significantly different between all sources after 2 weeks. With time, the soluble amounts of P increased to about 4 times at 4 weeks then dropped to about the values of the first pe- Element P K Ca Mg S Fe Mn Si Al Cr B Mo Concentration, % 0.55 0.08 32.1 5.40 0.06 19.5 2.32 7.02 1.32 0.06 0.02 ˂0.01 Table 1b: Total elements concentration in the basic slag sample Treatment P , µg g -1 Available form in: OSP 60.4 RP 21.6 BS 11.2 Compost 19.5 Humic acid 32.0 Mixtures (1:1, v/v) Citric acid 1 % + OSP (1:5) 133 Citric acid 1 % + RP (1:5) 47.0 Citric acid 1 % + BS (1:5) 30.7 EDTA 1 % + OSP (1:5) 125 EDTA 1 % + RP (1:5) 42.5 EDTA 1 % + BS (1:5) 32.0 Table 1a: Extractable- P in some of the studied treatments Acta agriculturae Slovenica, 117/3 – 2021 5 The possible use of scarce soluble materials as a source of phosphorus in Vicia faba L. grown in calcareous soils riod for the various P- sources. It also appears that the P solubility increased to about 5-6 times for all sources in the presence of the added bacteria compared to the treatments without adding P dissolving bacteria. With respect to the effect of the natural compounds (compost and humic acid), there was a slight increase in P solubility of the three sources when compost was added. Also, the same results were found when humic acid was added, but for OSP the P solubility increased more than the double. These results agreed with those obtained by Elhag et al. (2019) concerning the effect of humic acid on increasing P availability in washed sand. On the other hand, the effect of these natural organic sources was different when the dissolving bacteria was added, as the values of P solubility increased with com- post or humic acid in these sources with more P solu- bility for OSP than for RP or BS. This could be related to increasing the activity of the dissolving bacteria in the presence of organic sources. The bacteria decom- pose these compounds to simple materials beside the materials excreted by the bacteria. All these new com- pounds act in dissolving the P- sources. The effect was almost double in the OSP treatment than with RP or BS treatments. These results agreed with those obtained by Abd-Elrahman (2016) about the effect of P dissolv- ing bacteria on the solubility of P from OSP and RP fertilizers. Results of the ability of humic acid (HA) added or formed from the decomposition of compost or any simple or complex organic compounds resulted from the action of the bacteria added could be explained on the bases that these compounds may have positive or negative charges. The positive charges bind PO 4 3- groups, so help in releasing the phosphate from the in- soluble sources. On the other hand, the negative charge can bind Ca 2+ ion or any cation, so, also, helps in re- leasing the phosphate group from the insoluble sources (Campitelli et al., 2003). Regarding to the action of citric acid and EDTA, results show that in the absence of dissolving bacteria these compounds were superior to the compost and humic acid as they play their role directly without the action of the bacteria. This was clear with the insolu- ble sources RP and BS. The following descending or- der generally appeared in the chemically extractable mount of P with addition of solubilizing agents: citric acid > EDTA > HA > compost for these sources of P. Mihoub et al. (2018) reported that after a period of 960 h from incubation of highly calcareous soil samples (50 % CaCO 3 ) fertilized with triple superphosphate Particle size distribution, % Soluble cations, mmol c l -1 Sand 65.8 Ca 2+ 10.2 Silt 20.3 Mg 2+ 6.34 Clay 13.9 Na + 1.11 Textural class Sandy loam K + 0.52 FC, % 12.3 Soluble anions, mmol c l -1 W P, % 4.20 CO 3 2- n.d.* S P, % 31.5 HCO 3 - 4.27 CaCO 3 fractions, % Cl - 2.49 Coarse sand 16.8 SO 4 2- 6.28 Fine sand 8.30 Total macronutrients, % Silt 6.10 N 0.01 Clay 5.30 P 0.01 CaCO 3 , g kg -1 365 K 0.02 OM, g kg -1 1.10 Available macronutrients, µg g -1 CEC, cmol c kg -1 11.7 N 12.3 pH (1:2.5 soil:water suspension) 8.06 P 1.00 EC e , dS m -1 0.99 K 92.6 Table 2: Some initial physical and chemical characteristics of the surface layer of the experimental soil (0-20 cm) before plant cultivation *n.d. means not detected, field capacity (FC), wilting point (WP), saturation percent (SP), organic matter content in soil (OM), cation exchange capacity (CEC), and electrical conductivity of salts in soil extract (EC e ) Acta agriculturae Slovenica, 117/3 – 2021 6 A. M. ELGALA and S. H. ABD-ELRAHMAN and mono-ammonium phosphate and treated with cit- ric acid and oxalic acid solutions, treatments showed a significant decrease in extractable P with time, however, applying these solutions exerted a very favorable effect on P solubility in soil. Also, in our study, with the addi- tion of dissolving bacteria to the studied treatments ac- tivated the bacteria in dissolving the insoluble sources beside binding phosphate groups. Results also indicate that in the absence of dissolving bacteria, the soluble phosphates decrease by increasing the time of incuba- tion at 8 weeks. But in the presence of dissolving bac- teria the values in most treatments remain stable at 8 weeks. This clearly indicates that the compound formed in the presence of bacteria were more stable than that in the absence of bacteria. Abd-Elrahman (2016) re- ported that P dissolving bacteria increases P availability from OSP , and RP fertilizers added to a calcareous soil, with time. 3.3 POT EXPERIMENT 3.3.1 Available P in soil Table 4 shows the effect of different P sources and solubilizing agents on available P in calcareous soil in the presence or absence of P dissolving bacteria, during the physiological stages of faba bean growth. Results in- dicate that the values ranged from 1.2 to 10.4 µg g -1 in the absence of bacteria, and from 3.4 to 29.8 µg g -1 in the presence of bacteria. The solubility of P sources differs in their ability to release soluble P in the following order: OSP > RP > BS. In fact, this is related to their difference in their content of total- and extractable- P amounts. With respect to the ability of solubilizing agents in re- leasing P in the soil, it appears that they differ in the following descending order: citric acid > EDTA > HA > compost, similar to that found in the incubation experi- ment. As these agents were applied at the rate of 1 % (w/w), so it is expected that the active material of citric acid will be more than in compost and humic acid, as humus is composed of simple and complex compost as lignin (Taiwo, 2011). The superiority of citric acid com- pared to EDTA, could be explained on the basis that citric acid is smaller molecule compared to EDTA, so the active molecule well be more in 1 % of the material added compared to EDTA (Kanwal et al., 2014; Afshan et al., 2015). Besides the ability of EDTA to react with Ca to release P in the soil, it can chelate elements as Mg, Fe, Mn and Pb with higher stability (Hamed & Ga- mal, 2014; Kanwal et al., 2014). This may be the reason Treatment Available P in washed sand (µg g -1 ) Without adding bacteria In the presence of dissolving bacteria after 2 weeks 4 weeks 8 weeks after 2 weeks 4 weeks 8 weeks OSP 1.00 ± 0.03hi 4.20 ± 0.12hi 0.80 ± 0.04i 6.40 ± 0.11f 24.4 ± 1.92d 25.0 ± 2.14d OSP + Compost 1 % 1.80 ± 0.04g 5.80 ± 0.15f 1.60 ± 0.06g 7.60 ± 0.13d 24.6 ± 2.02cd 25.6 ± 2.19c OSP + Humic Acid 1 % 4.00 ± 0.14c 10.6 ± 0.26c 1.80 ± 0.06f 8.20 ± 0.16c 24.8 ± 2.03bc 25.0 ± 2.10d OSP + Citric Acid 1 % 9.40 ± 0.21a 12.8 ± 0.28a 13.2 ± 0.17a 13.8 ± 0.23a 54.6 ± 2.54a 57.6 ± 2.64a OSP + EDTA 1 % 6.80 ± 0.19b 11.0 ± 0.26c 4.60 ± 0.09b 11.4 ± 0.17b 25.0 ± 2.11b 27.4 ± 2.23b RP 0.80 ± 0.03i 3.80 ± 0.10ij 0.60 ± 0.05j 6.20 ± 0.10f 10.2 ± 0.21j 10.2 ± 0.19j RP + Compost 1 % 1.00 ± 0.03hi 5.20 ± 0.14g 1.40 ± 0.06h 6.40 ± 0.12f 11.4 ± 0.25h 10.8 ± 0.22hi RP + Humic Acid 1 % 1.60 ± 0.04g 5.60 ± 0.15f 1.40 ± 0.07h 6.80 ± 0.12e 12.2 ± 0.26f 12.6 ± 0.29f RP + Citric Acid 1 % 3.60 ± 0.11d 12.0 ± 0.27b 3.80 ± 0.08c 7.00 ± 0.13e 12.6 ± 0.27e 11.6 ± 0.23g RP + EDTA 1 % 3.00 ± 0.12e 6.80 ± 0.17e 3.60 ± 0.07d 7.60 ± 0.15d 11.8 ± 0.26g 13.0 ± 0.26e BS 0.40 ± 0.02j 3.40 ± 0.11j 0.40 ± 0.03k 4.40 ± 0.08k 8.60 ± 0.14k 9.00 ± 0.08k BS + Compost 1 % 1.00 ± 0.03hi 4.60 ± 0.13h 0.60 ± 0.04j 4.80 ± 0.08j 10.3 ± 0.22ij 10.6 ± 0.21i BS + Humic Acid 1 % 1.20 ± 0.03h 5.20 ± 0.15g 0.80 ± 0.06i 5.20 ± 0.09i 10.4 ± 0.22i 11.4 ± 0.23g BS + Citric Acid 1 % 3.40 ± 0.14d 9.60 ± 0.25d 3.60 ± 0.08d 5.40 ± 0.09hi 10.4 ± 0.24i 11.0 ± 0.22h BS + EDTA 1 % 2.60 ± 0.09f 6.60 ± 0.16e 3.40 ± 0.07e 5.60 ± 0.11h 10.2 ± 0.23j 10.8 ± 0.20hi Table 3: Effect of the studied treatments on chemically available P (µg g -1 ) in washed sand with time, in the presence or ab- sence of P- dissolving bacteria Ordinary Superphosphate (OSP), Rock Phosphate (RP), Basic Slag (BS). Values expressed as mean ± SE, the significant value was set at p ≤ 0.05. Different letters indicate significant difference between treatments. Acta agriculturae Slovenica, 117/3 – 2021 7 The possible use of scarce soluble materials as a source of phosphorus in Vicia faba L. grown in calcareous soils why extractable P from the treated soil with EDTA was less than citric acid. Mihoub et al. (2016) found that or- ganic acids, i.e., citric acid and oxalic acid, decreased P sorption capacity on the investigated calcareous soil whereas increased Gibbs free energy (ΔG) of P which reflected on increasing its solubility in soil, however, with citric acid more than oxalic acid. It appears that, as a function of time with growing the faba bean plants, extractable P decreased with time for the three P sources added alone and when compost and humic acid were added. This is related to activity of plant roots to utilize and withdraw the soluble P to fulfill the plant requirements. Also, it probably due to refixation of soluble P in soil by released Ca or another cation. In addition, data of extractable P in the presence of bacteria were significantly higher compared when bacteria were not added, and still the sequence was as follow: OSP > RP > BS. The reason for remaining BS in the last order may be due to its low P content. Yildirim & Prezzi (2011) reported that BS is containing small amounts of total P in the form of P 2 O 5 ranged from 0.01 to 3.3 % in all different types. Regarding the combinations between organic sub- stances and chelating agents, the treatment of HA+ cit- ric acid was superior in giving high extractable amount of P in soil, followed by HA+ EDTA in most physio- logical stages of growing bean plants, with significant differences in the presence of added bacteria. Humic acid plays a vital role in increasing P availability in soil (Doran et al., 2003; Sahin et al., 2014), plus its consider- able content of P (Table 1a). Citric acid, in addition to decrease soil pH, it makes complexes with Ca forming calcium citrate and releasing P in soluble form in the soil (Drouillon & Merckx, 2003). EDTA may solubilize the insoluble P forms in calcareous soils by chelating Ca 2+ and Mg 2+ cations, lowering soil pH and/ or the par- tial occupation of active anionic groups on the surface of CaCO 3 and clay minerals (Hamed & Gamal, 2014). It appears that the presence of bacteria played a protec- tive role against P- fixation (Abd-Elrahman, 2016; Płaza et al., 2021). 3.3.2 Vegetative growth parameters Data in Table 5 shows the effect of the studied P- treatments on faba bean plant height, and plant fresh and dry mass, in the presence or absence of P- dissolv- ing bacteria. The data of plant dry mass indicate that the addition of P- sources increased the dry mass yield more than the control and the rate of increase was in the same sequence mentioned for P availability in the soil: OSP > RP > BS (Table 4). The role of P in enhancing roots growth and their absorption efficiency in the soil was observed, which reflected on the plant growth and its yield. Razaq et al. (2017) found that applying P fer- tilizer increased root surface area, specific root length and root-shoot ratio. Fouda (2017) confirmed the effect of P fertilizer on increasing faba bean productivity. With respect to the effect of solubility agents, the sequence was different: EDTA > citric acid > HA > compost for OSP and RP , but the values were almost the same for BS. This indicates that despite relatively low total P content in BS (Table 1b), the P is found in a form easily released by the solubilizing agents. The addition of solubilizing bacteria increased the yield of dry mass to the extend to record slight significant difference be- tween the studied P- sources. This was also found when comparing the effect of solubilizing agents on yield in case of RP and BS were almost the same. Such soil with its high content of CaCO 3 is characterized by deficiency problems with some elements, particularly the micro- nutrients. The presence of appreciable amounts of Fe, Mn, Mg, S, B and other elements in BS had an effect of plant growth despite the low P content added (Table 1b). It is interesting to note that the highest yield re- corded for this experiment was when EDTA was added to OSP treatment, or mixed with HA, in the presence of P- dissolving bacteria. The above results indicate that solubilizing agents and dissolving bacteria not only act in solubilizing P from added materials and from soil, but also act in solubilizing other elements as Fe, Mn and Mg which are essential for plant growth. EDTA is known to chelate elements as Fe, Mn, Zn and Mg with higher stability as compared to citric acid or natural compounds as humic acid (Hamed & Gamal, 2014). Similar trend was observed with the other vegeta- tive growth parameters of faba bean plants as affected by the studied P treatments, with significant effect in the presence of P- dissolving bacteria as compared to not adding bacteria. Plant height ranged from 25 cm (in control treatment, without adding P sources and dissolving bacteria) to 70 cm (with applying the treat- ment of HA combined with EDTA, in the presence of dissolving bacteria). Also, the plant fresh mass ranged from 22.8 g plant -1 in control treatment (without any additions) to 60.5 g plant -1 with applying the treatment of HA combined with EDTA, in the presence of P- dis- solving bacteria. 3.3.3 Numbers of pods plant -1 , fresh mass of pods plant -1 and fresh mass of faba bean seeds plant -1 Results of mass of seeds (g plant -1 ) shown in Table Acta agriculturae Slovenica, 117/3 – 2021 8 A. M. ELGALA and S. H. ABD-ELRAHMAN Treatment Available P in soil (µg g -1 ) After seeds germination At the vegetative growth stage At the flowering stage At plant harvest (14 days after sowing) (60 days after sowing) (90 days after sowing) (145 days after sowing) (-PDB)* (+PDB)** (-PDB)* (+PDB)** (-PDB)* (+PDB)** (-PDB)* (+PDB)** Control (-P) 0.80 ± 0.04q 2.40 ± 0.12o 1.00 ± 0.04r 3.60 ± 0.26v 0.80 ± 0.04r 3.40 ± 0.18s 0.80 ± 0.03p 2.40 ± 0.12q OSP 2.64 ± 0.07k 6.40 ± 0.51h 4.20 ± 0.26jk 17.0 ± 1.16f 3.20 ± 0.13j 18.2 ± 1.26e 2.10 ± 0.08jk 9.20 ± 0.71l OSP + Compost 1 % 3.60 ± 0.10g 7.80 ± 0.80f 5.20 ± 0.34g 18.6 ± 1.21d 3.80 ± 0.15i 19.0 ± 1.33d 2.60 ± 0.09h 10.0 ± 0.83k OSP + Humic Acid 1 % 5.20 ± 0.23d 9.20 ± 0.86c 8.00 ± 0.62c 19.8 ± 1.28c 5.60 ± 0.34d 19.5 ± 1.35c 3.20 ± 0.17f 11.0 ± 0.92hi OSP + Citric Acid 1 % 9.83 ± 0.49a 15.6 ± 1.02a 10.4 ± 0.81a 29.0 ± 2.06a 10.2 ± 0.83a 29.8 ± 2.31a 4.80 ± 0.21a 18.4 ± 1.27a OSP + EDTA 1 % 7.80 ± 0.33b 12.4 ± 0.97b 8.80 ± 0.64b 21.2 ± 1.78b 8.20 ± 0.60b 22.0 ± 2.01b 4.20 ± 0.20c 13.6 ± 1.16c RP 2.00 ± 0.06m 5.60 ± 0.29i 3.80 ± 0.15l 10.6 ± 0.84mn 2.40 ± 0.11n 10.7 ± 0.80m 1.40 ± 0.07n 10.0 ± 0.85k RP + Compost 1 % 2.41 ± 0.09k 6.80 ± 0.48g 4.00 ± 0.23kl 11.4 ± 0.90l 2.44 ± 0.12n 11.2 ± 0.92kl 2.01 ± 0.09k 10.8 ± 0.89ij RP + Humic Acid 1 % 3.20 ± 0.12h 8.00 ± 0.85ef 5.20 ± 0.30g 12.2 ± 0.95k 4.00 ± 0.23h 12.8 ± 0.96j 2.40 ± 0.09i 11.6 ± 0.91g RP + Citric Acid 1 % 5.60 ± 0.26c 8.41 ± 0.84d 6.80 ± 0.42d 13.4 ± 0.98j 6.40 ± 0.41c 13.6 ± 0.098i 4.40 ± 0.23b 13.4 ± 1.15cd RP + EDTA 1 % 4.20 ± 0.18f 8.20 ± 0.80de 6.40 ± 0.40e 12.5 ± 0.93k 4.80 ± 0.20f 12.8 ± 0.95j 4.03 ± 0.21c 12.8 ± 1.06e BS 1.80 ± 0.09n 4.40 ± 0.23l 3.20 ± 0.17n 8.23 ± 0.61s 2.00 ± 0.10o 8.40 ± 0.61q 1.00 ± 0.06o 9.20 ± 0.72l BS + Compost 1 % 2.20 ± 0.10l 5.20 ± 0.28j 4.00 ± 0.22kl 9.20 ± 0.74q 2.40 ± 0.12n 9.00 ± 0.73op 1.61 ± 0.07m 10.6 ± 0.85j BS + Humic Acid 1 % 2.80 ± 0.13j 6.20 ± 0.47i 4.63 ± 0.25hi 9.60 ± 0.76p 3.00 ± 0.16k 10.0 ± 0.80n 1.80 ± 0.07l 12.2 ± 1.02f BS + Citric Acid 1 % 4.60 ± 0.17e 6.80 ± 0.50g 5.80 ± 0.35f 10.8 ± 0.81m 5.20 ± 0.29e 11.6 ± 0.94k 3.80 ± 0.17d 13.2 ± 1.11de BS + EDTA 1 % 3.60 ± 0.15g 6.40 ± 0.49h 4.86 ± 0.26h 10.4 ± 0.80n 4.20 ± 0.23g 10.8 ± 0.84lm 3.61 ± 0.16e 12.6 ± 1.06e OSP+ RP+ BS (50 % for everyone) 2.40 ± 0.11k 7.02 ± 0.68g 4.40 ± 0.24ij 18.0 ± 1.27e 3.20 ± 0.16j 18.8 ± 1.20d 1.80 ± 0.08l 9.00 ± 0.78l Compost 1 % 1.80 ± 0.07n 4.60 ± 0.28kl 2.40 ± 0.08p 8.81 ± 0.62r 2.40 ± 0.13n 8.60 ± 0.67pq 2.00 ± 0.09k 4.82 ± 0.21o Humic Acid 1 % 2.20 ± 0.013l 5.60 ± 0.30i 3.20 ± 0.18n 10.0 ± 0.81o 3.00 ± 0.15k 10.2 ± 0.83n 2.20 ± 0.09j 5.70 ± 0.23n Citric Acid 1 % 1.40 ± 0.08o 3.80 ± 0.19m 1.80 ± 0.06q 7.40 ± 0.54t 2.00 ± 0.11o 7.80 ± 0.55r 2.00 ± 0.08k 4.80 ± 0.22o EDTA 1 % 1.20 ± 0.06p 3.40 ± 0.17n 1.60 ± 0.06q 6.80 ± 0.44u 1.60 ± 0.08q 7.40 ± 0.53r 1.42 ± 0.07n 4.00 ± 0.19p Compost 1 %+ Humic Acid 1 % (50 % for both) 2.20 ± 0.10l 5.20 ± 0.26j 2.80 ± 0.09o 14.6 ± 1.10h 2.60 ± 0.13m 14.7 ± 1.15hi 2.20 ± 0.10j 11.1 ± 0.90h Citric Acid 1 %+ EDTA 1 % (50 % for both) 1.40 ± 0.07o 3.60 ± 0.18mn 1.89 ± 0.07q 9.87 ± 0.71op 1.80 ± 0.08p 9.20 ± 0.72o 1.60 ± 0.08mn 6.60 ± 0.42m Compost 1 %+ Citric Acid 1 % (50 % for both) 2.20 ± 0.12l 5.20 ± 0.29j 3.00 ± 0.23no 15.0 ± 1.18j 2.80 ± 0.15l 15.8 ± 1.24f 2.60 ± 0.12h 10.8 ± 0.89ij Compost 1 %+ EDTA 1 % (50 % for both) 2.02 ± 0.09m 4.80 ± 0.25k 3.00 ± 0.25no 14.0 ± 1.05i 2.60 ± 0.14m 14.6 ± 1.13hi 2.60 ± 0.11h 10.0 ± 0.90k Humic Acid 1 %+ Citric Acid 1 % (50 % for both) 3.00 ± 0.15i 6.60 ± 0.54h 4.02 ± 0.26kl 17.0 ± 1.17f 3.80 ± 0.17i 19.2 ± 1.38cd 3.00 ± 0.18g 15.6 ± 1.18b Humic Acid 1 %+ EDTA 1 % (50 % for both) 2.40 ± 0.11k 5.40 ± 0.32i 3.40 ± 0.25m 14.2 ± 1.12i 3.00 ± 0.18k 15.2 ± 1.20g 2.65 ± 0.13h 11.2 ± 0.97h Table 4: Effect of the applied P sources on chemically available P (µg g -1 ) in El-Nubaria calcareous soil, in the presence or absence of P- dissolving bacteria, during the physi- ological stages of growing faba bean plants *(-PDB) means without adding P dissolving bacteria, **(+PDB) means in the presence of P dissolving bacteria. Ordinary Superphosphate (OSP), Rock Phosphate (RP), Basic Slag (BS). Values ex- pressed as mean ± SE, the significant value was set at p ≤ 0.05. Different letters indicate significant difference between treatments. Acta agriculturae Slovenica, 117/3 – 2021 9 The possible use of scarce soluble materials as a source of phosphorus in Vicia faba L. grown in calcareous soils Table 5: Effect of the applied P sources on vegetative growth parameters of faba bean plants cultivated on El-Nubaria calcareous soil, in the presence or absence of P- dis- solving bacteria *(-PDB) means without adding P dissolving bacteria, **(+PDB) means in the presence of P dissolving bacteria. Ordinary Superphosphate (OSP), Rock Phosphate (RP), Basic Slag (BS). Values ex- pressed as mean ± SE, the significant value was set at p ≤ 0.05. Different letters indicate significant difference between treatments Treatments Plant height, cm Plant fresh mass, g Plant dry mass, g (-PDB)* (+PDB)** (-PDB)* (+PDB)** (-PDB)* (+PDB)** Control (-P) 25.0 ± 2.21q 35.5 ± 2.44s 22.8 ± 2.13s 28.4 ± 2.38u 4.94 ± 0.23s 7.48 ± 0.54s OSP 32.5 ± 2.80k 39.0 ± 2.68o 26.2 ± 2.25m 34.9 ± 3.29o 7.12 ± 0.50l 10.9 ± 0.81no OSP + Compost 1 % 34.0 ± 2.87i 44.0 ± 3.15j 28.1 ± 2.31j 37.8 ± 3.40l 8.34 ± 0.57k 12.1 ± 0.98k OSP + Humic Acid 1 % 36.3 ± 3.01g 47.0 ± 3.21g 29.8 ± 2.45i 41.7 ± 4.04ij 9.18 ± 0.65gh 13.5 ± 1.07h OSP + Citric Acid 1 % 50.0 ± 4.43b 58.4 ± 4.01d 33.3 ± 2.70e 49.1 ± 4.25d 9.90 ± 0.72d 16.1 ± 1.29c OSP + EDTA 1 % 53.0 ± 4.50a 60.1 ± 4.29c 34.6 ± 2.76c 58.6 ± 4.46b 11.3 ± 0.86a 18.0 ± 1.33b RP 28.0 ± 2.52o 36.5 ± 2.35r 23.4 ± 2.22q 32.2 ± 2.89qrs 6.69 ± 0.43p 9.83 ± 0.76r RP + Compost 1 % 30.5 ± 3.22l 40.7 ± 2.80n 27.1 ± 2.29l 35.4 ± 2.93n 7.19 ± 0.52l 11.1 ± 0.91no RP + Humic Acid 1 % 32.0 ± 2.34k 43.0 ± 3.39k 28.3 ± 2.36j 38.5 ± 3.45k 8.90 ± 0.64i 11.4 ± 0.90m RP + Citric Acid 1 % 40.0 ± 3.11f 46.0 ± 3.50h 31.8 ± 2.83g 45.5 ± 4.11f 9.30 ± 0.73f 14.8 ± 1.12f RP + EDTA 1 % 41.0 ± 3.23e 48.0 ± 3.55f 32.3 ± 2.87f 47.8 ± 4.19e 9.82 ± 0.79d 15.8 ± 1.18d BS 26.3 ± 2.25p 35.9 ± 2.37rs 23.1 ± 2.26r 30.9 ± 2.47t 5.93 ± 0.32r 9.65 ± 0.76r BS + Compost 1 % 29.1 ± 2.53n 38.0 ± 2.41pq 24.1 ± 2.32o 32.5 ± 2.90qr 6.89 ± 0.39no 10.4 ± 0.87pq BS + Humic Acid 1 % 30.5 ± 2.87l 38.2 ± 2.38p 24.4 ± 2.34n 32.7 ± 2.89p 6.93 ± 0.45mno 10.5 ± 0.88p BS + Citric Acid 1 % 36.5 ± 2.91g 43.5 ± 3.60jk 27.6 ± 2.78k 37.7 ± 3.37l 8.90 ± 0.62i 11.8 ± 0.94l BS + EDTA 1 % 35.4 ± 2.76h 45.0 ± 3.62i 27.8 ± 2.76k 41.9 ± 4.12i 9.11 ± 0.71h 12.6 ± 0.98ij OSP+ RP+ BS (50 % for everyone) 32.0 ± 2.79k 41.3 ± 3.22lm 27.2 ± 2.69l 37.1 ± 3.32m 8.63 ± 0.59j 12.5 ± 0.97j Compost 1 % 28.0 ± 2.23o 37.8 ± 2.43q 23.4 ± 2.23q 32.0 ± 3.45s 6.33 ± 0.44q 10.3 ± 0.86q Humic Acid 1 % 30.0 ± 2.69m 38.0 ± 2.47pq 23.9 ± 2.29p 32.6 ± 3.51pq 6.81 ± 0.47o 10.4 ± 0.87pq Citric Acid 1 % 33.4 ± 2.80j 41.1 ± 3.26mn 24.0 ± 2.38op 33.1 ± 3.60p 6.97 ± 0.48mn 10.8 ± 0.92o EDTA 1 % 34.0 ± 2.85i 41.7 ± 3.35l 24.0 ± 2.37op 35.4 ± 3.66n 6.98 ± 0.51m 11.9 ± 0.98kl Compost 1 %+ Humic Acid 1 % (50 % for both) 35.4 ± 2.49h 47.0 ± 3.56g 30.5 ± 2.44h 41.3 ± 4.13j 9.23 ± 0.42fg 12.7 ± 1.02i Citric Acid 1 %+ EDTA 1 % (50 % for both) 42.5 ± 3.44d 45.0 ± 3.41i 34.1 ± 2.51d 44.0 ± 4.20g 10.1 ± 0.78c 14.8 ± 1.13f Compost 1 %+ Citric Acid 1 % (50 % for both) 40.0 ± 3.39f 54.3 ± 4.52e 32.3 ± 2.49f 43.1 ± 4.18h 9.10 ± 0.69h 12.9 ± 1.05i Compost 1 %+ EDTA 1 % (50 % for both) 42.0 ± 3.38d 60.0 ± 4.58c 32.5 ± 2.47f 48.7 ± 4.70d 9.31 ± 0.73f 14.3 ± 1.08g Humic Acid 1 %+ Citric Acid 1 % (50 % for both) 46.0 ± 2.56c 64.0 ± 4.71b 36.1 ± 2.72b 53.9 ± 4.93c 9.50 ± 0.75e 15.3 ± 1.17e Humic Acid 1 %+ EDTA 1 % (50 % for both) 50.0 ± 2.70b 70.0 ± 4.93a 39.4 ± 3.14a 60.5 ± 5.03a 10.9 ± 0.82b 18.7 ± 1.38a Acta agriculturae Slovenica, 117/3 – 2021 10 A. M. ELGALA and S. H. ABD-ELRAHMAN 6 indicates the values ranged between 1.21 g to 11.5 g with lower values for treatments without bacteria addi- tion. The addition of P fertilizers significantly increased the seed yield by about 5 times with OSP and only about 3 times for RP and BS compared to the control without bacteria addition. When solubilizing bacteria was added the magnitude of increase was only about the double in case of OSP and slightly less than double in case of RP and BS. This means that the solubilizing bacteria play a major role than the P- sources. Bacillus megaterium var. phosphaticum produced acids that en- hanced the availability of phosphates and increased the uptake of other nutrients, leading to increased yields (Cakmakci et al., 1999; Saxena et al., 2020; Płaza et al., 2021). With respect to solubilizing agents, there are slight significant difference among solubilizing agents in the absence of bacteria. However, when solubilizing bacte- ria was added the effect follows the sequence: EDTA > citric acid > HA > compost in case of OSP and RP, but the effect was almost the same when BS was used. This means there are factors other than P in BS contributed to the production of seeds yield. It is possible that the presence of appreciable amounts of Mg, S, Fe, Mn, B and other elements played a role in plant growth, al- though P percentage was relatively low compared to RP and OSP (Yildirim & Prezzi, 2011). Regarding the interaction among the studied treat- ments, the treatment of HA+ EDTA gave the highest seeds yield, recording 8.57 g plant -1 without adding bac- teria. While recorded 11.5 g plant -1 in the presence of added bacteria. Similar trend was found with the other yield parameters, with high significant difference in the presence of P- dissolving bacteria. Number of pods plant -1 varied from 1 in control treatment (without any additions) to 6.67 that recorded by many treatments, OSP in combination with citric acid or EDTA and the treatment of HA combined with EDTA, in the presence of P- dissolving bacteria. Regarding the fresh weight of pods, ranged from 2.59 g plant -1 (in control treatment, without any additions) to 22.3 g plant -1 with applying the treatment of HA combined with EDTA, in the pres- ence of P- dissolving bacteria (Table 6). 3.3.4 N, P and K concentrations in faba bean leaves Results of P concentration in leaves of faba bean plants (Table 7) indicated that by addition of P sources, P contents increased with remarkable increase with applying OSP treatment than applying RP or BS treat- ments. This was found in case of without adding or with adding solubilizing bacteria, but the values were generally higher with the later. Elhag et al. (2019) re- ported that, increasing available P in soil by addition of P sources was reflected on increasing P concentration in bean roots and shoots, with high concentrations in shoots more than roots. However, no remarkable difference in concentra- tion among solubilizing agents, except when EDTA or citric acid was added with BS. This again clearly indi- cate the role of these relatively small compounds in sol- ubilizing not only insoluble P, but also other elements as Mg, Fe, Mn in BS which may activate plant roots to absorb nutrients. The enhanced role of EDTA or citric acid in calcareous soils is not only due to acidification of the plant rhizosphere, but also to its Ca and Mg com- plexing capacity (Drouillon & Merckx, 2003; Hamed & Gamal, 2014). Mihoub et al. (2019) found that P uptake by wheat plants grown in alkaline calcareous soil was 0.493 mg P pot -1 in the control treatment, however, it reached 0.701 and 0.785 mg P pot -1 in the amended pots with pigeon manure juice and citric acid, respectively. Regarding the interaction between solubilizing agents, the treatment of HA+ EDTA, followed by that plus citric acid gave the highest concentration of P in plant leaves, with significant difference as compared to the other treatments. Sahin et al. (2014) found that hu- mic substances in interaction with P in the soil could decrease the P- fixation and increase the P- uptake by plants. With respect to the effect of the studied treatments on N and K concentrations in plant leaves (Table 7), it was clear that the addition of P increased N and K content in all treatments. Also, there was a remarkable difference recorded with respect to the solubilizing agents and bacterial additions. Although HA and com- post enhanced the plant uptake from N and K, due to their considerable content of the macro elements; the treatments of EDTA and citric acid were superior in in- creasing plant uptake of both. These may be due to en- hancing root efficiency in absorbing nutrients from soil and added fertilizers, lowering soil pH, and their high Ca and Mg complexing capacity (Drouillon & Merckx, 2003; Hamed & Gamal, 2014). The main problem of the investigated soil is its high content of CaCO 3 (36.5 %, Table 2), and this is in- fluence on nutrients availability and uptake by plants. So, the soil moisture content should be kept up to field capacity till the end of the experimental work. Also, the role of EDTA and citric acid in enhancing root growth and absorption, besides their interaction with CaCO 3 in soil as well as their effects on lowering soil pH; re- flected on enhancing plant growth and productivity, as compared to the effect of compost or HA (Campitelli et al., 2003; Drouillon & Merckx, 2003). The interac- Acta agriculturae Slovenica, 117/3 – 2021 11 The possible use of scarce soluble materials as a source of phosphorus in Vicia faba L. grown in calcareous soils Table 6: Numbers of pods plant -1 , fresh mass of pods plant -1 and fresh mass of faba bean seeds plant -1 cultivated on El-Nubaria calcareous soil as affected by the different P sources, in the presence or absence of P- dissolving bacteria *(-PDB) means without adding P dissolving bacteria, **(+PDB) means in the presence of P dissolving bacteria. Ordinary Superphosphate (OSP), Rock Phosphate (RP), Basic Slag (BS). Values ex- pressed as mean ± SE, the significant value was set at p ≤ 0.05. Different letters indicate significant difference between treatments Treatments No. of pods plant -1 F. mass of pods (g plant -1 ) F. mass of seeds (g plant -1 ) (-PDB)* (+PDB)** (-PDB)* (+PDB)** (-PDB)* (+PDB)** Control (-P) 1.00 ± 0.03k 2.00 ± 0.05l 2.59 ± 0.10r 5.93 ± 0.33t 1.21 ± 0.05q 2.58 ± 0.09p OSP 3.00 ± 0.07g 5.00 ± 0.27e 7.42 ± 0.50m 11.6 ± 0.90n 4.19 ± 0.12k 6.43 ± 0.39i OSP + Compost 1 % 3.67 ± 0.10e 6.00 ± 0.30c 8.23 ± 0.62j 13.7 ± 0.98j 4.56 ± 0.18h 7.61 ± 0.47g OSP + Humic Acid 1 % 4.00 ± 0.10d 6.33 ± 0.30b 8.55 ± 0.65i 15.7 ± 1.02h 4.72 ± 0.20g 8.06 ± 0.49f OSP + Citric Acid 1 % 4.33 ± 0.13c 6.67 ± 0.33a 9.72 ± 0.70h 17.8 ± 1.13f 5.23 ± 0.24e 9.78 ± 0.56d OSP + EDTA 1 % 4.67 ± 0.17b 6.67 ± 0.35a 9.84 ± 0.74g 19.9 ± 1.29c 5.50 ± 0.27d 10.1 ± 0.70c RP 2.33 ± 0.05i 4.00 ± 0.25h 6.87 ± 0.43o 10.1 ± 0.81q 3.53 ± 0.08o 5.11 ± 0.21o RP + Compost 1 % 2.67 ± 0.07h 4.33 ± 0.30g 6.92 ± 0.47o 10.3 ± 0.82q 3.87 ± 0.09m 5.56 ± 0.23m RP + Humic Acid 1 % 3.00 ± 0.07g 4.67 ± 0.30f 7.61 ± 0.52l 12.6 ± 0.86l 4.31 ± 0.14j 6.53 ± 0.30i RP + Citric Acid 1 % 3.67 ± 0.10e 5.00 ± 0.33e 7.98 ± 0.53jk 13.6 ± 0.94j 4.43 ± 0.16i 7.32 ± 0.43h RP + EDTA 1 % 3.67 ± 0.10e 5.33 ± 0.35d 9.76 ± 0.73gh 14.8 ± 1.05i 5.42 ± 0.21d 8.12 ± 0.49f BS 2.00 ± 0.05j 3.00 ± 0.20k 6.10 ± 0.43q 9.38 ± 0.70s 3.51 ± 0.08o 5.17 ± 0.21n BS + Compost 1 % 2.67 ± 0.07h 3.67 ± 0.25i 6.55 ± 0.45p 10.1 ± 0.82q 3.75 ± 0.10n 5.15 ± 0.20n BS + Humic Acid 1 % 3.00 ± 0.10g 4.00 ± 0.30h 7.27 ± 0.57n 11.5 ± 0.93n 4.07 ± 0.13l 6.20 ± 0.33k BS + Citric Acid 1 % 3.00 ± 0.07g 4.33 ± 0.35g 7.88 ± 0.56k 12.2 ± 0.96m 4.39 ± 0.15ij 6.41 ± 0.35ij BS + EDTA 1 % 3.67 ± 0.13e 4.67 ± 0.30f 8.11 ± 0.60j 12.5 ± 0.97l 4.58 ± 0.16h 7.30 ± 0.41h OSP+ RP+ BS (50 % for everyone) 4.33 ± 0.20c 6.00 ± 0.37c 9.76 ± 0.74gh 13.1 ± 1.04k 5.45 ± 0.25d 7.60 ± 0.49g Compost 1 % 2.33 ± 0.07i 3.33 ± 0.17j 6.50 ± 0.45p 9.81 ± 0.72r 3.23 ± 0.10p 5.13 ± 0.24no Humic Acid 1 % 2.67 ± 0.10h 4.00 ± 0.30h 6.67 ± 0.46p 10.3 ± 0.85q 3.86 ± 0.12m 5.47 ± 0.27m Citric Acid 1 % 3.00 ± 0.07g 4.33 ± 0.30g 7.01 ± 0.49o 10.7 ± 0.87p 3.98 ± 0.15l 5.83 ± 0.30l EDTA 1 % 3.33 ± 0.15f 4.33 ± 0.25g 7.29 ± 0.51mn 11.1 ± 0.95o 4.03 ± 0.15l 6.25 ± 0.38ijk Compost 1 %+ Humic Acid 1 % (50 % for both) 4.33 ± 0.20c 5.00 ± 0.23e 10.6 ± 0.78d 16.0 ± 1.15g 5.27 ± 0.23e 8.53 ± 0.46e Citric Acid 1 %+ EDTA 1 % (50 % for both) 5.00 ± 0.27a 6.00 ± 0.27c 13.7 ± 0.99b 19.5 ± 1.28d 7.15 ± 0.45b 10.3 ± 0.53c Compost 1 %+ Citric Acid 1 % (50 % for both) 4.00 ± 0.20d 6.00 ± 0.30c 10.1 ± 0.80f 18.8 ± 1.16e 5.08 ± 0.28f 9.51 ± 0.49d Compost 1 %+ EDTA 1 % (50 % for both) 4.00 ± 0.23d 6.33 ± 0.30b 10.4 ± 0.87e 19.7 ± 1.27cd 5.31 ± 0.29e 10.2 ± 0.54c Humic Acid 1 %+ Citric Acid 1 % (50 % for both) 4.67 ± 0.25b 6.33 ± 0.35b 12.2 ± 0.91c 21.0 ± 1.33b 6.94 ± 0.32c 11.2 ± 0.63b Humic Acid 1 %+ EDTA 1 % (50 % for both) 5.00 ± 0.27a 6.67 ± 0.37a 16.8 ± 1.12a 22.3 ± 1.35a 8.57 ± 0.50a 11.5 ± 0.64a Acta agriculturae Slovenica, 117/3 – 2021 12 A. M. ELGALA and S. H. ABD-ELRAHMAN Table 7: N, P and K concentrations in faba bean leaves cultivated on El-Nubaria calcareous soil as affected by the different P sources, in the presence or absence of P- dis- solving bacteria *(-PDB) means without adding P dissolving bacteria, **(+PDB) means in the presence of P dissolving bacteria. Ordinary Superphosphate (OSP), Rock Phosphate (RP), Basic Slag (BS). Values ex- pressed as mean ± SE, the significant value was set at p ≤ 0.05. Different letters indicate significant difference between treatments Treatments N, % P, % K, % (-PDB)* (+PDB)** (-PDB)* (+PDB)** (-PDB)* (+PDB)** Control (-P) 2.10 ± 0.07s 2.34 ± 0.09t 0.16 ± 0.02m 0.22 ± 0.03p 1.69 ± 0.08r 1.95 ± 0.08v OSP 2.39 ± 0.09o 2.81 ± 0.12o 0.27 ± 0.05h 0.34 ± 0.09j 2.04 ± 0.10kl 2.16 ± 0.12q OSP + Compost 1 % 2.43 ± 0.09jkl 2.92 ± 0.14l 0.30 ± 0.07e 0.37 ± 0.09g 2.13 ± 0.10i 2.27 ± 0.23jkl OSP + Humic Acid 1 % 2.49 ± 0.10h 2.97 ± 0.15hi 0.33 ± 0.07c 0.39 ± 0.08e 2.17 ± 0.12g 2.31 ± 0.22i OSP + Citric Acid 1 % 2.65 ± 0.12c 3.11 ± 0.18f 0.37 ± 0.08b 0.42 ± 0.10c 2.21 ± 0.15e 2.39 ± 0.28g OSP + EDTA 1 % 2.72 ± 0.13b 3.26 ± 0.21e 0.39 ± 0.08a 0.46 ± 0.14a 2.30 ± 0.19b 2.45 ± 0.30f RP 2.31 ± 0.09q 2.74 ± 0.10r 0.24 ± 0.04j 0.30 ± 0.06m 1.92 ± 0.09p 2.11 ± 0.09s RP + Compost 1 % 2.40 ± 0.10no 2.84 ± 0.10n 0.27 ± 0.05h 0.32 ± 0.06l 2.03 ± 0.09l 2.18 ± 0.10op RP + Humic Acid 1 % 2.46 ± 0.10i 2.88 ± 0.12m 0.28 ± 0.05g 0.35 ± 0.06i 2.12 ± 0.10ij 2.25 ± 0.18l RP + Citric Acid 1 % 2.50 ± 0.12gh 2.96 ± 0.13ij 0.30 ± 0.07e 0.37 ± 0.07g 2.17 ± 0.13g 2.28 ± 0.20j RP + EDTA 1 % 2.59 ± 0.13d 3.09 ± 0.15g 0.31 ± 0.07d 0.40 ± 0.07d 2.25 ± 0.18c 2.32 ± 0.21i BS 2.28 ± 0.08r 2.69 ± 0.10s 0.21 ± 0.03l 0.26 ± 0.04o 1.87 ± 0.08q 2.08 ± 0.10t BS + Compost 1 % 2.36 ± 0.09p 2.75 ± 0.11qr 0.24 ± 0.04j 0.30 ± 0.07m 1.97 ± 0.09n 2.14 ± 0.11r BS + Humic Acid 1 % 2.41 ± 0.10mn 2.76 ± 0.11q 0.26 ± 0.06i 0.33 ± 0.07k 2.01 ± 0.09m 2.19 ± 0.11o BS + Citric Acid 1 % 2.45 ± 0.10ij 2.87 ± 0.12m 0.28 ± 0.07g 0.36 ± 0.08h 2.13 ± 0.11i 2.23 ± 0.15m BS + EDTA 1 % 2.55 ± 0.12e 2.98 ± 0.14h 0.29 ± 0.07f 0.39 ± 0.09e 2.21 ± 0.16e 2.26 ± 0.15kl OSP+ RP+ BS (50 % for everyone) 2.35 ± 0.08p 2.83 ± 0.12n 0.30 ± 0.08e 0.35 ± 0.06i 2.05 ± 0.10k 2.05 ± 0.11u Compost 1 % 2.32 ± 0.08q 2.70 ± 0.11s 0.22 ± 0.04k 0.28 ± 0.03n 1.94 ± 0.09o 2.10 ± 0.12t Humic Acid 1 % 2.39 ± 0.09o 2.74 ± 0.11r 0.24 ± 0.04j 0.29 ± 0.03n 1.98 ± 0.09n 2.16 ± 0.14q Citric Acid 1 % 2.42 ± 0.11lm 2.81 ± 0.12o 0.27 ± 0.05h 0.32 ± 0.07l 2.11 ± 0.012j 2.17 ± 0.15pq EDTA 1 % 2.51 ± 0.12fg 2.94 ± 0.15k 0.28 ± 0.07g 0.34 ± 0.08j 2.16 ± 0.13gh 2.21 ± 0.18n Compost 1 %+ Humic Acid 1 % (50 % for both) 2.43 ± 0.12jkl 2.79 ± 0.12p 0.26 ± 0.06i 0.32 ± 0.07l 2.15 ± 0.15h 2.36 ± 0.23h Citric Acid 1 %+ EDTA 1 % (50 % for both) 2.55 ± 0.13e 3.28 ± 0.19d 0.28 ± 0.08g 0.38 ± 0.08f 2.23 ± 0.19d 2.48 ± 0.26e Compost 1 %+ Citric Acid 1 % (50 % for both) 2.52 ± 0.14f 3.27 ± 0.20de 0.27 ± 0.06h 0.39 ± 0.09e 2.19 ± 0.17f 2.59 ± 0.27d Compost 1 %+ EDTA 1 % (50 % for both) 2.56 ± 0.13c 3.36 ± 0.22c 0.28 ± 0.08g 0.40 ± 0.10d 2.21 ± 0.19e 2.67 ± 0.30c Humic Acid 1 %+ Citric Acid 1 % (50 % for both) 2.74 ± 0.18b 3.45 ± 0.25b 0.28 ± 0.09g 0.43 ± 0.11b 2.29 ± 0.21b 2.81 ± 0.30b Humic Acid 1 %+ EDTA 1 % (50 % for both) 2.90 ± 0.19a 3.58 ± 0.26a 0.30 ± 0.08e 0.46 ± 0.12a 2.34 ± 0.25a 2.94 ± 0.33a Acta agriculturae Slovenica, 117/3 – 2021 13 The possible use of scarce soluble materials as a source of phosphorus in Vicia faba L. grown in calcareous soils tion among the studied treatments gave better results, especially between chelating agents and organic com- pounds. In addition, the P dissolving bacteria produce organic and inorganic acids that mobilize P and other nutrients and encourage plant growth, as well as releas- ing phosphatase enzymes to mineralize organic P (Il- lmer et al., 1995; Cakmakci et al., 1999; Amalraj et al., 2012; Płaza et al., 2021). 4 CONCLUSION Many factors are affecting the solubility of P in cal- careous soils. In our study we tried to use some sources of P as well as organic substances and chelating agents, the interaction between them being the best. BS gave promising results especially when combined with citric acid and EDTA not only in calcareous soil, but possibly in soils poor in nutrient elements as sandy soils, even under the alkaline conditions. Using BS gained many benefits such as recycling of wastes, protecting the en- vironment from contamination, and being a source of P fertilizer. Also, application of citric acid and EDTA enhanced faba bean growth in the investigated soil, par- ticularly with addition of organic substances. In addi- tion, adding solubilizing bacteria played a major role than the P- sources in enhancing the availability of P and increasing the uptake of other nutrients, leading to increased yield. 5 RECOMMENDATIONS The use of RP as a source of P in calcareous soil at the time of applying the organic and bio fertilizers and close to plant roots is so beneficial. Applying RP or BS during the preparation of compost will enrich the compost with P. Application of BS along with organic fertilizers and chelating agents can be recommended in low fertile soil as sandy soil. 6 REFERENCES Abd-Elrahman, Shaimaa H. (2016). Effect of unconventional phosphorus sources and phosphate solubilizing bacte- ria on fractions of phosphorus in a calcareous soil cul- tivated with wheat plants. 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