P-solubilizing Bacteria as a Panacea to Alleviate Stress Effects of High Soil CaCO3 content in Phaseolus Vulgaris with Special Reference to P-Releasing Enzymes

Purpose: The present study examines the role of leguminous compost (LC), humic acid (HA), and phosphate-solubilizing bacteria (P-SB) in alleviating the stress effects of high soil CaCO 3 content in Phaseolus vulgaris. Methods: Two pot trials for two consecutive seasons; fall 2019 and summer 2020 were implemented in an open greenhouse. With four replicates specied for each of ve treatments, a randomized complete plot design was assigned to each trial. Results: Inoculation of calcareous soil with P-SB (a 1: 1 mixture of two Pseudomonas sp.; Ps. mallei and Ps. cepaceae) signicantly exceeded LC, HA, or even LC+HA for the positive results obtained. P-SB facilitated nutrient solubility (e.g., N, K, Fe, and Mn), including conversion of insoluble phosphorous into a form available in the tested soil due to increased soil enzymatic activities (e.g., phosphatases and phytases). This mechanism, combined with a decrease in soil calcium carbonate content and an increase in cation exchange capacity (CEC) and organic matter (OM) content, increased the availability of various nutrients to plants, including P, in the soil, which contributed to the increased plant output. Adequate P content in plants led to a marked decrease in plant acid phosphatase activity under high content of CaCO 3 . Conclusions The study concluded that the use of P-SB promotes biological activities, nutrient availability, and thus the productivity of calcareous soils, enabling Phaseolus vulgaris plants to withstand stress produced by high CaCO 3 content through the development and/or adoption of potentially effective mechanisms.


Introduction
The availability of essential nutrients for plant production, especially phosphorus (P), in defective agricultural lands such as calcareous soils, is very important due to the globally large areas of these lands (Rady et al. 2020). Frequent nutrient applications are required to achieve high crop yields if effective tools are not used to address soil problems, especially nutrient xation. Like essential nutrients, P is a prime nutrient for plant performance, but unlike N, there is no sizeable atmospheric source that helps provide it biologically (Khan et al. 2009;Ezawa et al. 2002;). As a base nutrient with metabolic, structural, and functional properties, P in its available state is critical to the performance of the plant (Stauffer and Sulewski 2004). A great amount of total P is insoluble in the soil, and therefore cannot be absorbed by plant roots, which leads to its de ciency that limits crop productivity globally (Arcand and Schneider 2006;Yang et al. 2012). The availability of P, especially in calcareous soils, is largely controlled by rates of immobilization and mineralization as biological-mediated processes (Zou et al. 1992). Unlike N, P supply is not easily replenished, so it is necessary to preferably utilize P reserves and rectify chemically bound P (Cordell et al. 2009). Thus, it is quickly restricted into unavailable forms resulting in lower P utilization e ciency regardless of the amount applied to soil (Nautiyal et al. 2000). In the showed that the remarkable availability of N, P, and K can be obtained with the application of organic conditioner (Abou Hussien 2020).
Studies aimed at selecting bacteria capable of dissolving and mineralizing soil P have been carried out to boost the sustainable development of agriculture. This can be achieved by striving to minimize the use of chemical fertilizers and favoring the development of ecologically balanced agricultural environments (Matos 2017). Many soil microorganisms can dissolve unavailable forms of P bound to Ca by organic acids excreted through metabolic activities. These organic acids either dissolve rock phosphate or chelate Ca ions to release P into soil solution (Nautiyal et al. 2000). There is strong evidence that many soil bacteria can convert P into a form available to plants (Khan et al. 2009). Since the middle of the last century and possibly earlier, phosphate-solubilizing bacteria (P-SB) have been used as bio-fertilizers (Kudashev 1956;Krasilinikov 1957). P-SB play an important role in converting insoluble P into a form more available to plants (Sharon et al. 2016). A wide range of microbial species; bacteria, fungi, actinomycetes, and even algae play a base role in solubilizing P, but bacteria are the largest use because they are most effective at dissolving P. Microorganisms secrete organic acids to solubilize P complexes (Goldstein 1995;Sharon et al. 2016) and/or chelate cations, which bind to P ions (PO 4 3− ) to release P (Vyas and Gulati 2009). Several bacteria can solubilize phosphate, amongst them the Pseudomonas sp. (Verma et al. 2001;Garg et al. 2001), which are found in a large number in biological environments and can solubilize the metallic P complexes and release the bioavailable form of P (Rady et al. 2020). Mechanisms by which microorganisms act to solubilize P include the release of organic acid anions, siderophores, protons, hydroxyl anions, CO 2 , and extracellular enzymes or biochemical P mineralization, and release of P during substrate degradation (McGill and Cole 1981). This promotes soil fertility and increases the availability of nutrients including P, thus shortening the period of repair of low-quality soil (Shi et al. 2017). Extensive studies have been implemented to isolate P-SB from different plant rhizospheres (Chung et al. 2005;Acevedo et al. 2014;Wu et al. 2014;Anzuay et al. 2015).
Compared with leguminous compost (LC), humic acid (HA), and humi ed compost (HA-LC), very little research has investigated the impact of P-SB on nutrient recycling, especially P, after their application to calcareous soils. The present study investigates the potential positive impact of inoculating calcareous soil (19.6% CaCO 3 ) with P-SB compared to the application of the tested soil with LC, HA, or HA-LC on Phaseolus vulgaris plant growth, yield, nutrient contents, including P, and acid phosphatase activity. Soil physicochemical properties, including soil nutrient contents and P-solubilizing enzyme activities, were also investigated. Where Phaseolus vulgaris is a crop sensitive to different stress types (Sultana et al. 2014;Bargaz et al. 2016), including calcareous state stress (Rady et al. 2020), was selected for this study. Based on health, color, and size, the standard Bronco seed cultivar of common beans (Phaseolus vulgaris L.) was secured from Agricultural Research Center (Horticulture Research Institute), Egypt. Sodium hypochlorite solution (1%) was used to sterilize the seed surface for 5 min. Then, distilled water was used to wash the seeds thoroughly several times to exclude the residue of the sterilization solution. After drying in the air for 1 h, the seeds were prepared for sowing using plastic pots with a diameter of 36 cm and a depth of 30 cm. A weight of 12 kg calcareous soil with 19.6% CaCO 3 was allocated to each pot.
Based on the methods detailed in Page et al. (1982) and Klute and Dirksen (1986), soil chemical and physical properties were analyzed and are shown in Table 1. Available Zn 2.10 ± 0.13 "dS m − 1 " means decisiemens per meter, "CEC" means cation exchange capacity, "cmol c kg − 1 " means centimole of cation exchange capacity per kilogram soil, and "mg kg − 1 " means milligram per kilogram.
For the fall season 2019, ve treatments each with four replicates (5 pots for each replicate); A total of 100 pots were assigned to this study. The calcareous soil of 20 pots was left without any supplementation and identi ed as a control. A mixture of Pseudomonas cepaceae and Ps. mallei identi ed as phosphate-solubilizing bacteria (P-SB) was used to inoculate the soil of another 20 pots. The leguminous compost (LC; 10 g kg − 1 soil) and humic acid (90.3% net HA; 50 mg kg − 1 soil) were added and mixed well with the calcareous soil of 20 pots for each. A humi ed-compost (HA-LC) was added at a rate of 5 g kg − 1 to the soil of the remaining 20 pots. HA-LC was prepared by adding 50 g HA to 2.5 kg LC and mixed well. Before applying the investigated treatments, the soil of each pot (12 kg) was fertilized with 1.2, 2.4, and 3.6 g of potassium sulfate; 48% K 2 O, calcium superphosphate; 15% P 2 O 5 , and ammonium sulfate; 20% N. These treatments were repeated for summer 2020 using the same soil as fall 2019.
Using a randomized complete plot design, the experimental treatments were arranged using 20 pots with four replicates each. Rotation (from place to place) was performed daily for pots of all treatments to ensure fairness in sunlight intensity and light distribution. Ten homogeneous seeds were planted in each pot. After full emergence, only three standard seedlings per pot were maintained by successful thinning. Plants of all treatments were watered daily, plus all necessary agricultural practices were applied as recommended to produce Phaseolus vulgaris commercially.
At 48 days after sowing (DAS) and after harvesting, soil samples were collected randomly from 3 pots in each treatment of each growing season to assess the changes in soil properties and soil enzymatic activities. At 48 DAS, plants (n = 9) were harvested for growth evaluation; Weights of fresh and dry shoot for each plant. At harvesting, green pod yield and dry seed yield were assessed in the remaining pots.

Preparation of leguminous compost
Green faba bean shoots (2.50 kg) were mixed with different organic materials such as bulking agents (50 g), potassium humate (100 g), and N-sources such as Egyptian clover plants (1.25 kg) and cattle manure (1.25 g). The proportions speci ed for the mixtures of the compost were 48% for faba bean shoots, 25% for Egyptian clover, 25% for cattle manure, and 2% for potassium humate. All these mixtures were mixed well for composting in a pilot-plant using the system of turning-pile in trapezoidal piles (the base dimensions were 2 × 0.75 × 0.50 m in length, width, and height, respectively). From May to September the piles were turned every 2 weeks during the bio-oxidative phase. Moisture and temperature were monitored during the composting process. While turning the piles, the moisture level was kept in the range of 40-60% by adding water. The analysis of the obtained compost was as follows: 19.6%, 7.5, 2.1 dS m − 1 , 115 g kg − 1 , 33 g kg − 1 , and 152 g kg − 1 for organic matter content, pH, EC, N, P, and K, respectively.

Phosphate-solubilizing bacteria (P-SB) isolation and identi cation
Pseudomonas cepaceae and Ps. mallei were obtained with the help of Nutrient Broth medium (NB). These bacteria were isolated from the plant rhizosphere and identi ed molecularly in the National Research Center, Egypt. The PCR technique was implemented to identify bacteria using the following oligonucleotide primers: Target species; Ps. mallei and Ps. Cepaceae, Primers; CVP 23 − 2 and M 23 − 2, 23S rDNA helices containing target position; 78ab and 78ab, Sequence; 5'-CAC CGA AAC TAG CG-3' and 5'-CAC CGA AAC TAG CA-3', Size of PCR product (bp); 526 and 526, and Annealing temperature; 47 and 47°C, respectively. The bacteria (Ps. cepaceae and Ps. mallei) were tested for their capability of P solubilization and pH reduction. They were identi ed as P-SB and plant growth-promoting rhizobacteria.
Besides, the two bacterial isolates had no anti-activity against one another.

Preparation and application of P-SB
A mixture of a 1: 1 ratio of compost and peat has functioned as a carrier for the P-SB inoculant. Using aluminum foil, this carrier was encapsulated and sterilized with an autoclave. Then, the carrier was provided with 10% P-SB inoculant, this is, each 10 kg carrier was enriched with 1 L of inoculant. The P-SB inoculant was used or was packed and stored in a drying place until use. For P-SB treatments, calcareous soil was inoculated with bacterial inoculant at 1 g (0.1 mL net P-SB) kg -1 of soil 48 h before sowing.

Soil enzyme activity assay
Samples of the tested soil were collected 48 DAS, as well as at harvest (the end of the experiment), and then the replications were mixed well to clean by passing through a < 2-mm sieve. Assaying the phosphatase activity was performed colorimetrically based on the procedures of Guan (1986). Besides, phytase activity was assayed in suspensions and solutions of soil against a 20 mM acidi ed InsP6 substrate applying procedures of George et al. (2005) and Giaveno et al. (2010). Then, the concentration of P was determined by applying the procedures of Irving and McLaughlin (1990). As P released during 1 h assaying, calculation of phytase activity was performed as nKat g − 1 soil using the following equation:

Assessments of soil properties
From each treatment, soil samples were collected 48 DAS, as well as at harvest from random three pots to assess organic matter (%), CaCO 3 (%), cation exchange capacity, and nutrient; P, N, K, Fe, and Mn contents (Klute and Dirksen 1986;Page et al. 1982).

Growth and yield determinations
Plant shoots were sampled 48 DAS for the fresh weight (n = 9), as well as for dry weight after oven-drying at 70°C until constant weights were obtained. In the green pods marketing stage (62-70 DAS), six plants were used for picking green pods to assess pod weight (g) and total green pods per plant (g). For the dry yield, the remaining 80-day-old plants were used, the pods were picked and left for air-drying for 3 d. Next, the dry pods were used to evaluate dry seeds' weight per plant (g).

Determination of leaf contents of nutrients
Powdered dry leaf samples from all investigated treatments were used to determine nutrient contents. Total N was assessed using procedures depending on the micro-Kjeldahl technique. P was assessed colorimetrically using stannous chloride-ammonium molybdate reagent (King 1951), after its extraction by sodium bicarbonate (Olsen 1954). K + was assessed using a ame photometer (ELE Flame Photometer, Leighton Buzzard, UK). Fe 2+ , Mn 2+ , Zn 2+ , and Cu 2+ contents were determined by atomic absorption spectrophotometry (Chapman 1965).

Acid phosphatase activity assay
To extract the enzyme, sodium acetate-acetic acid buffer at 20 ml was used to grind 1.0 g of fresh material from plant leaves and roots. The extract centrifugation was practiced for 10 min (30,000 ×g, 2°C). The acid phosphatase activity was assayed in the supernatant according to Basford's procedures (1979). Assaying the activity of acid phosphatase enzyme was guided by p-nitrophenol as a standard curve according to Clark (1975).

Statistical analysis
The procedures of Levene (1961) and Shapiro and Wilk (1965) were used to test the homogeneity of error variances and the normality distribution, respectively. Next, all data underwent two (plant analyses) or three analyses of variance (soil analyses). The difference between every two means was signi cant at P ≤ 0.05 with the use of Tukey's HSD test (honestly signi cant difference). The analysis was implemented statistically with the help of GenStat 17th Ed. (VSN Int. Ltd, Hemel Hempstead, UK).

Soil characterization
The chemical and physical characterization of the investigated calcareous soil data as presented in Table 1 indicates that the textural class is clay (clay percentage exceeds 50%, silt is about 29 and sand is about 20%), pH is more than 8.15 which indicates that the soil is alkaline, salinity is somewhat low within the range of 2.3 dS m − 1 , organic matter (OM) content is low (about 0.54%), calcium carbonate is high and exceeds 19% which indicate that the soil is calcareous as per Leytem and Mikkelsen (2005), who de ned calcareous soil as contains 14-17% or more calcium carbonate content. Cation exchange capacity (CEC) is low (about 5.82 cmol c kg − 1 ). The available N, P, and K as macronutrients and Fe, Mn, and Zn as micronutrients are also included. The soil is classi ed following the USDA norms and standards as Typic Haplotorrerts (Abdelfattah 2007;Soil Survey Staff 2014;Shahid et al. 2014).

Effects of the different treatments on soil and plant parameters
The resulted data of the effects of the growing season, sampling time, and soil application with leguminous compost (LC), humic acid (HA), humi ed compost (HA-LC), or phosphate-solubilizing bacteria (P-SB) on soil and plant parameters including soil enzyme activities, soil properties (available P, OM, CaCO 3 , CEC), soil and plant nutrient contents, plant growth and yield, and acid phosphatase activity in plant leaves and roots, are summarized below (Tables 2-6). "**" signi cant at P ≤ 0.01 and "*" signi cant at P ≤ 0.05. Data presented are means ± SE (n = 9). Different letters next to mean values indicate signi cant differences at P ≤ 0.05.

Soil enzymatic activities
For the growing season, phosphatase and phytase activities were signi cantly increased in soil samples taken in the summer season, 2020 by 83.2 and 73.0%, respectively, compared to their activities in soil samples gathered in the fall season, 2019 (Table 2). Regarding sampling time, soil samples collected after plant harvesting awarded signi cant increases of 19.5 and 18.4% for phosphatase and phytase activities, respectively in comparison with those of soil samples gathered at 45 days after sowing. Concerning soil treatments, all the soil applications; LC, HA, HA-LC, or P-SB signi cantly increased phosphatase and phytase activities compared to control. The best soil treatment was P-SB that signi cantly exceeded all the other treatments (e.g., LC, HA, and HA-LC) and conferred 256.9 and 221.6%, respectively compared to control. As the main factors showed signi cant (P ≤ 0.05) or highly signi cant (P ≤ 0.01) differences, all interactions between/among the tested factors were signi cant (P ≤ 0.05).
32.2 Soil properties (available P, OM, CaCO 3 , and CEC) For the growing season, the available phosphorous, organic matter, and CEC were signi cantly increased in soil samples collected in summer season 2020 compared with those collected in fall 2019 by 122.92, 28.95, 80.14%, respectively (

Growth and yield
For the growing season, the weight of fresh and dry shoot for each plant, and the weight of green pods and dry seeds per plant increased signi cantly in plant samples collected in summer 2020 compared with the ones collected in fall 2019 by 25.64, 24.47, 14.22, and 17.79%, respectively (Table 5). Concerning soil treatments, all of the soil applications including LC, HA, HA-LC, or P-SB signi cantly increased the fresh and dry weights of plant shoots but recorded a highly signi cant increase for the weights of green pods and dry seeds per plant, compared with the control. P-SB recorded the best soil treatment that signi cantly exceeded all the other treatments (e.g., LC, HA, and HA-LC) and conferred 134.01% for shoot fresh weight plant − 1 , 158.33% for shoot dry weight plant − 1 , 555.08% for green pods weight plant − 1 , and 709.29% for dry seeds weight plant − 1 compared to the control (Table 5). As the main factors showed signi cant (P ≤ 0.05) or highly signi cant (P ≤ 0.01) differences, all interactions between/among the tested factors were signi cant (P ≤ 0.05). Signi cance S × STR * * * * "**" signi cant at P ≤ 0.01 and "*" signi cant at P ≤ 0.05. Data presented are means ± SE (n = 9). Different letters next to mean values indicate signi cant differences at P ≤ 0.05.

Activity of acid phosphatase enzyme
For the growing season, the activity of the phosphatase enzyme of leaves and roots was signi cantly decreased in plant samples collected in summer 2020 compared with the ones collected in fall 2019 by 10.58 and 9.11%, respectively (Table 6). For soil treatments, all the soil applications including LC, HA, HA-LC, and P-SB markedly suppressed the activity of phosphatase enzyme of leaves and roots compared with the control. However, P-SB was the best soil treatment, and signi cantly surpassed all the other treatments (e.g., LC, HA, and HA-LC) and conferred 61.64% (of leaves) and 64.32% (of roots) for the phosphatase activity compared with the control (Table 6). As the main factors showed signi cant (P ≤ 0.05) or highly signi cant (P ≤ 0.01) differences, all interactions between/among the tested factors were signi cant (P ≤ 0.05). Signi cance S × STR * * "**" signi cant at P ≤ 0.01 and "*" signi cant at P ≤ 0.05. Data presented are means ± SE (n = 9). Different letters next to mean values indicate signi cant differences at P ≤ 0.05.

Leaf macro-nutrient contents
For the growing season, N, P, and K contents were markedly elevated in plant samples collected in summer 2020 compared with those collected in fall 2019 by 17.05, 16.22, and 17.73%, respectively (Table 7). Concerning soil treatments, all the soil applications including LC, HA, HA-LC, and P-SB markedly elevated N, P, and K contents compared with the control. However, P-SB was the best soil treatment that signi cantly surpassed all the other treatments (e.g., LC, HA, and HA-LC) and conferred 87.25, 292.5, and 17.36% for the N, P, and K contents respectively, compared to the control (Table 7). As main factors were showed signi cant (P ≤ 0.05) or highly signi cant (P ≤ 0.01) differences, interactions between/among the growing season and soil treatments for P and K were signi cant (P ≤ 0.05), but not signi cant for N. Signi cance S × STR Ns * * "**" signi cant at P ≤ 0.01, "*" signi cant at P ≤ 0.05, and "ns" non-signi cant. Data presented are means ± SE (n = 9). Different letters next to mean values indicate signi cant differences at P ≤ 0.05.

Discussion
Nutrients are very important for different crop plants to provide optimum growth and productivity. Among them, P is the main nutrient with key functional roles. It helps legume plants x N and positively affects plant growth and development. It is also an essential constituent of many biomolecules (e.g., DNA, RNA, ATP, and phospholipids), in addition to its role in stabilizing the thylakoid membrane needful for chlorophyll molecule biosynthesis and positioning (Schachtman et al. 1998;Rodríguez and Fraga, 1999;Rady et al. 2020). However, there is an ongoing problem related to nutrients, especially P with calcareous soils. The calcareous soil tested in the current study has undesirable properties, poor structure, low fertility, and nutritional imbalance. It also has a high CaCO 3 content and a high pH value, along with a low cation exchange capacity (CEC) and organic matter (OM) content, thus low available nutrient contents (Table 12). These unwanted characteristics always accompany less productive or unproductive soils (Aboukila et al. 2018;Rady et al. 2020). Thus, Phaseolus vulgaris, as a crop sensitive to various stressors become an unproductive crop when grown in such soils (Sultana et al. 2014;Bargaz et al. 2016), including high CaCO 3 content (Rady et al. 2020). Thus, effective tools must be applied to reform the harsh conditions of the soil tested in this study and make insoluble nutrients (including P) soluble, and available to plants.
The research strategy pursued in this study is to use four tools (e.g., humic acid; HA, leguminous compost; LC, humi ed compost; HA + LC, and phosphate-solubilizing bacteria; P-SB) to apply them to the tested calcareous soil (19.6% CaCO 3 ). They all succeeded in releasing the nutrients, especially P, to be available for uptake by the plant, but the treatment of inoculating the soil with P-SB was the best.
By adding OM such as HA or compost to defective (calcareous) soil, it tends to repair the soil (Aboukila et al. 2018;Belal et al. 2019;Manirakiza and Şeker 2020) by improving its physical (e.g., soil water retention capacity, rate of in ltration, and particle aggregation), chemical (e.g., nutrients, CaCO 3 , EC e , pH, CEC, and OM), and biological (e.g., microorganisms) characteristics (Brady and Weil 2008). Many characteristics [e.g., nutrients (P, N, K, Fe, and Mn), CEC, OM, CaCO 3 , enzyme (phosphatase and phytase) activities] that were tested in this study were markedly improved with HA or LC application to the soil compared to those obtained with the control (Tables 2, 3, 4 and 9). These positive soil outcomes contributed to a marked decrease in leaf and root acid phosphatase activity (due to the increase in P content that meets the need of the plant), and a considerable increase in Phaseolus vulgaris plant growth, nutrient contents (e.g., N, K, Fe, Mn, Zn, and Cu), especially P and green pods and dry seeds yields (Tables 4, 5, 6 and 9).
Increased yields under the stress of high soil CaCO 3 content may be due to the bene cial in uence of HA on ameliorating growth and activation of biochemical processes (e.g., photosynthesis, chlorophyll content, and respiration) of plants (Hegazi 2004), which contribute to all yields of Phaseolus vulgaris plants (Tables 3, 5 and 9). These positive ndings obtained with HA on defective soil are in parallel with those of Brady and Weil (2008), and Belal et al. (2019). Brady and Weil (2008) stated, in general, that humus as a colloid containing cations of the essential nutrients in a readily exchangeable form exempli es 50-90% of the capacity to uptake cations in the mineral topsoil. Seyedbagheri (2010) using calcareous soils, stated that HA improves the organic-clay complexes reactions, which contribute to the formation of stable humus that ameliorates the physical, chemical, and biological functions of these soils. In the soil, HA helps cover clay domains with various active organic acids that have been liberated from HA. Then, these clay domains form coarse aqueous-stable aggregates segregated by a coarse pore procedure, which increases the permeability of the soil thus helping to easily leach the excess soluble salts to diminish the ECe value (Belal et al. 2019). HA can increase soil biological activity (bene cial bacteria), which can e ciently contribute to restoring calcareous soils. The increase in bacterial activities by HA leads to produce certain organic acids and plant hormones (e.g., cytokinins and indole acetic acid).
The hormones induce the roots and root hair proliferation to raise nutrient-absorbing surfaces. Also, the organic acids solubilize organic and inorganic forms of bene cial elements (especially P), thus increasing plant growth and different yields (Abou Zied et al. 2005;Belal et al. 2019). The perceived increase in the nutrient contents available in calcareous soil tested in this study with HA application may be due to the observed increase in soil CEC, CaCO 3 , and OM contents, as well as enzyme (phosphatase and phytase) activities that help increase available P (Tables 2, 3 and 9). When added to the calcareous soil, HA improves soil biology conditions, which encourage easy release and mobility of nutrients into the soil in forms more available to plants (Mahmoud et al. 2011). Belal et al. (2019) attributed the improvement of biological activity of the calcareous soil with HA treatment to bioactive substances released to promote the nutrient solubility in the soil from both its native and additive sources and to keep these solubilized nutrients in forms more available to plants. The promoted impact of HA on phytonutrient contents (Tables 3, 4 and 9) may be attributed to improved root system development (David et al. 1994) and boosted cell plasma membrane permeability (Ulukan 2008). The effect of a greater improvement of smaller molecular sizes of HA on uptake of plant nitrates (Calvo et al. 2014) comes from their transfer into the cell plasma membrane, where they e ciently affect nutrient assimilations (Quilty and Cattle 2011). Khaled and Fawy (2011) also reported that HA may interact with the structures of phospholipids in cell plasma membranes as a nutrient carrier, demonstrating anti-stress impacts under different conditions of abiotic stresses (Kulikova et al. 2005), such as the high CaCO 3 content (19.6%) under study.
Similar to our ndings (Tables 3, 4, and 9), Aboukila et al. (2018) and Manirakiza and Şeker (2020) indicated that calcareous soils treated with compost display a marked rise in the OM and nutrient; Zn, Mn, Fe, and K contents due to the compost's high content of these OM and nutrients, which are subsequently released into the soil through bacterial decomposition (Fischer and Glaser 2012). Manirakiza and Şeker (2020) reported increased soil nutrient and OM contents, which they attributed to the richness of the compost in nutrients and organic carbon. Ghosh et al. (2015) and Naeem et al. (2018) showed a rise in the soil contents of OM and N compounds after composting because of the compost's richness in organic carbon and N and the acceleration of ammoni cation and nitri cation rates after excretion of exudates from plant roots. The content of N compounds has also been reported to increase after adding compost to reduce leaching (Yao et al. 2012). Like our data (Tables 2-6, and 9), the available P increases signi cantly after adding compost to the soil. The compost's richness in available P that is liberated from the compost into the soil through a process called "mineralization" could explain this nding (Aboukila et al. 2018;Manirakiza and Şeker 2020). Also, the sorption of Fe 3+ , Al 3+ , Ca 2+ , Mg 2+ , and K + , especially acidic cations (Fe 3+ and Al 3+ ), after adding the compost increases the available P in the soil solution (Xu et al. 2014). This study presents an increase in available P attributable to low CaCO 3 content, and high OM content, CEC, and phosphatase and phytase activities in soil, which helped increase the solubility of P along with other nutrients after LC addition (Tables 2-6 and 9).
A synergistic a rmative in uence on nutrient contents of plants has been reported after adding compost to soil (Liu et al. 2012). Our ndings (Tables 2-5 and 9) are supported by Manirakiza and Şeker (2020) who reported enhanced plant growth traits due to soil treatment with compost, which can be explained by improved soil structure, fertility, and water retention after the release of nutrients from applied compost, and the synergism among nutrients and increased their retention (Sohi et al. 2009). This study presents a higher pH (8.15 ± 0.41; Table 1) of the tested calcareous soil which falls outside the recommended range for optimal nutrient availability, and thus the nutrient availability for plants is very low (Jones and Jacobsen 2005). However, in this study, compost use increased nutrient availability and uptake, which increased the nutrient contents (e.g., Cu, Zn, Mn, Fe, K, and N), especially P, in the plant (Tables 3, 4 and 9) and was positively re ected in the Phaseolus vulgaris growth and yield components ( Table 5). The ndings of Doan et al. (2015) and Manirakiza and Şeker (2020) are similar to ours. As demonstrated by this study (Tables 3, 4 and 9), the use of compost resulted in a marked rise in plant content of P and other nutrients (Cu, Zn, Mn, Fe, K, and N), which is due to improved soil fertility (Manirakiza and Şeker 2020). In calcareous soils, the signi cant binding of Ca-P decreased P availability and uptake, and thus decreased the P content in plants as demonstrated with the control in this study (Tables 3-5 and 9). However, the use of compost signi cantly increased the availability and uptake of P and P content and other nutrient contents in plants, which may be due to improved soil fertility (Tables 2-6 and 9). Jones and Jacobsen (2005) indicated that the capacity of nutrient uptake depends on the density of the root system and the nutrient content in the soil solution. Agegnehu et al. (2016) pointed out that nutrient uptake is enhanced in maize plants because of raised nutrient availability by lowering the soil pH after application of compost. In calcareous soils, P is presented as a critical factor, like other essential nutrients, for plant performance. Compost increases the uptake of nutrients (Mn, Cu, Zn, Fe, Ca, K, P, and N) by crop plants grown in calcareous soil, and indicates that nutrient solubility is likely attributable to plant root-secreted organic compound, which promotes the availability of nutrients to plants (Nur et al. 2014;Inal et al. 2015).
Application of calcareous soil with HA + LC signi cantly exceeded both HA and LC applied alone for the investigated soil properties, growth and different yields of common bean plants and the plant content of different nutrients, especially P (Tables 2-6 and 9). These signi cant ndings from HA + LC treatment compared to HA and LC separately applied are attributed to the synergistic and positive integrative effects of both HA and LC as elucidated above.
The treatment of soil inoculation with phosphate-solubilizing bacteria (P-SB; Pseudomonas cepaceae and Pseudomonas mallei) signi cantly exceeded all other treatments (HA, LC, and HA + LC) for the examined soil properties, growth, and different yields of Phaseolus vulgaris plants and the plant content of different nutrients, especially P (Tables 2-6 and 9).
To release P in a bioavailable form (orthophosphate), the metallic P complex can be solubilized by Pseudomonas sp. through many speci c mechanisms. The production of organic acids, Fe-siderophore, and soil enzymes such as phosphatase and phytase are some of these bacterial mechanisms, which are base functions in solubilizing the organic forms of P so that they are absorbable to plant roots (Rady et al. 2020).
In this study, P release in favor of plant roots could easily be achieved by inoculating the tested calcareous soil by P-SB, which effectively increased soil phytase and phosphatase activities, CEC, OM, available nutrients, and greatly reduced the soil pH value and CaCO 3 content. Thus, P-SB can make unwanted calcareous soils productive.
In the calcareous soil tested in this study, P-SB (a mixture from Pseudomonas cepaceae and Ps. mallei) simpli ed the conversion of insoluble P to be available to Phaseolus vulgaris plants, a mechanism that contributed to the increased P content in the plant, which in turn contributed to increasing plant productivity (Tables 4, 5 and 9). The ndings of Shi et al. (2017) and Rady et al. (2020) con rm the results of this study. This enhanced effect of P-SB strains was due to their effective phospholysis (P release) ability through the increased phytase and phosphatase enzyme activities in the soil as an e cient mechanism, resulting in increased availability of P to plant roots (Tables 2 and 9). The data of this study indicate that inoculation of calcareous soil with P-SB is a key determinant of its fertility. This positive nding can be elucidated based on higher available nutrients, including P, and OM, as well as lower CaCO 3 content obtained by P-SB treatment (Tables 3 and 9). These positive results were re ected in higher growth and different yield components of common bean plants (Tables 3 and 9).
Synergistically, Pseudomonas sp. work on the production of phosphatases (Tables 2 and 9) by some processes (e.g., immobilization and mineralization) to convert organic P into inorganic form throughout the plant life cycle, so that Pseudomonas sp. growth can be optimized continuously (Fitriatin et al. 2014). Another effective mechanism, various organic acids are both qualitatively and quantitatively secreted, mainly as a gene dependent, in soil by P-SB strains (Zhen et al. 2016). These organic acids compete with P ions for P adsorption sites, resulting in higher P release in favor of plants. P-SB enhance the calcareous soil productivity and increase its capacity for microorganisms, phytase and phosphatase enzyme activities (biological activity), and nutrient contents including available P (biochemical activity) in this soil (Tables 2, 3 and 9).
P-SB enhance P utilization e ciency through direct and/or indirect way. The direct way is organic acids exudation and P-hydrolyzing phosphatase enzymes to improve P pool bioavailability. The indirect one is the production of certain hormones, and toxin-resistance, and antifungal compounds, as well as other biologically active molecules. They can all support a robust shoot/root system set up under environmental constraints (Shi et al. 2017), including increasing the CaCO 3 content of the soil under study. Organic acids contribute to solubilizing P often by decreasing the soil pH value and chelating cation properties, as shown in inoculation of calcareous soil with P-SB (Rady et al. 2020). Behera et al.
(2017) stated that acidi cation of the microbial cell perimeter, as a potential mechanism, induces P anion release by substituting H + and Ca 2+ . Alori et al. (2017) reported some other conceivable mechanisms for P solubilization in calcareous soil including proton release after NH 4 assimilation by microbial cells, production of H 2 SO 4 and HNO 3 (inorganic acids), and speci c enzymes (Tables 2 and 9), which act on amphiphilic fatty substances. Along with the microbial solubility of P, microorganisms also mineralize the organo-P, playing a major role in cycling the P to be available to plants. Alori et al. (2017) added that Preleasing enzymes (phytases and phosphatases) produced by P-SB broadly control the mineralization of P. Besides, other features deserve agricultural attention such as the production of plant hormones and antifungal compounds, and regulation of the main pathways included in plant metabolism to enhance the ability of plants to withstand environmental stresses (Sharma et al. 2013).
The increased availability of nutrients, including P, through P-SB application to the soil enhanced the performance and nutrient content (including P) of Phaseolus vulgaris plants. This allowed Phaseolus vulgaris to possess the advantage of staying green (data are not shown), increasing the seed lling period under stress. This nding is obtained due to the plant's ability to e ciently uptake nutrients from calcareous soil (Tables 4 and 9). This allows plants to ful ll meristematic activities including cell expansions due to adequate provision of water against stress resulting from the increase in soil CaCO 3 content under study. The worthy increase in the content of K + ion (Tables 4 and 9) acted in its ionic state as a powerful osmoprotectant. Recently, Rady et al. (2020) reported that increased solubilized P in calcareous soil due to its inoculation with P-SB (Tables 2 and 9) is re ected positively in the P content of Phaseolus vulgaris plants (Tables 5 and 9). This report (Rady et al. 2020) added that the plants' high nutritional content-enabled them to have a potent antioxidant defense system against the harsh conditions of a high CaCO 3 state.
In this study, inoculation of calcareous soil with P-SB helps to provide plants with enough P, decreasing root, and leaf acid phosphatase activity (Tables 2 and 9). This nding can be attributed to the P content that is reached to meet plant needs. The ndings of Rady et al. (2020) con rm the ndings of this study, indicating that increased plant content of P induces decreases in acid phosphatase activities in common bean leaves and roots. The authors attributed this nding to that when the soil contains su cient available P (with P-SB) for uptake by plants, it restricts acid phosphatase activity in the plant and increases P mineralization in the soil. Additionally, phosphatase activity in plant root system tends to increase along with a decrease in shoot P content, and under P de ciency, the activity of root and shoot phosphatase increases (Romer and Fahning 1998;Kaya 2002). Eligible plants, with su cient P through the application of P-SB, with several potential mechanisms to be developed and/or adopted to boost their tolerance to stress induced by high CaCO 3 content. For instance, the high plant K + ion content confers an osmoprotectant mechanism against water loss to keep su cient leaf water content to help the plant perform well under the harsh conditions of high CaCO 3 stress.
The results obtained in the summer season of 2020 signi cantly exceeded the results obtained in the fall season of 2019 in terms of soil properties, growth and yields of Phaseolus vulgaris plants, and plant content of different nutrients, especially P (Tables 2-6 and 9). This may be attributed to the same soil used in the summer of 2020 for the fall season of 2019, which awarded an opportunity to release excess nutrients from the soil due to the increased decomposition of LC and HA added in the previous season, in addition to the more solubility of P and other nutrients that occurred by P-SB.

Conclusions
This study shows that inoculating the calcareous soil with phosphate-solubilizing bacteria (a 1: 1 mixture of two Pseudomonas sp.; Ps. mallei and Ps. cepaceae) markedly exceeded the soil treatment with humic acid, leguminous compost, or humic acid + leguminous compost in enhancing the growth and productions of common bean plants under stress induced by high soil calcium carbonate (CaCO 3 ) content. Phosphate-solubilizing bacteria facilitated the solubility of phosphorus (P) and other nutrients (e.g., Mn, Fe, K, and N) by increasing the enzymatic activities of the soil (e.g., phosphatase and phytase), along with an increase in the soil cation exchange capacity and organic matter content along with a lower CaCO 3 content, resulting in augmented nutrient availability in the soil for plant roots. This led to adequate P content in Phaseolus vulgaris plant, leading to a marked decrease in acid phosphatase activity in plant leaves and roots. P-mediated growth promotion under high CaCO 3 stress was attributed to the improvement of soil biological activities, phytase and phosphatase activities, available nutrient contents including P; mechanisms by which phosphate-solubilizing bacteria enabled Phaseolus vulgaris plants to boost their tolerance to the stress of high CaCO 3 content. Therefore, future research works into this concern will be useful.

Declarations
Compliance with Ethical Standards Con ict of Interest The authors declare that they have no con ict of interest. Funding