Phosphorus Availability in Wheat , in Volcanic Soils Inoculated with Phosphate-Solubilizing Bacillus thuringiensis

The use of phosphate solubilizing bacteria (PSB) is an ecological strategy that allows for increasing the availability of phosphorus (P) in soil. The objective of this study was to evaluate P availability in wheat, in soils derived from volcanic ash (Andisol and Ultisol,) and inoculated with phosphate-solubilizing Bacillus thuringiensis, the experiment was conducted in pots under greenhouse conditions using a completely randomized design. Wheat plants were inoculated and re-inoculated at 20 and 46 days after sowing (DAS), respectively, with B. thuringiensis; and, soil and plant sampling was performed after 46, 66, and 87 days based on the Zadoks growth scale (Z). The inoculation resulted in an 11% increase in P of the rhizosphere at Z46 (Ultisol), P also increased 34% and 67% in aerial tissues at Z46 (Andisol and Ultisol), respectively, while an increase of 75% was observed in root tissues at Z87 (Ultisol). Similarly, the inoculation resulted in increases in acid phosphatase activity (Andisol), soil microbial biomass (Andisol and Ultisol), and root biomass in plants (Ultisol), without achieving increase of the aerial biomass of the plants. The phosphate solubilizing B. thuringiensis strain showed some positive, but also negative effects in soils and plants, depending on the soil.


Introduction
Phosphorus (P) is one of the most important chemical element for plant growth and development; in fact, it forms part of the ATP molecules and plays a decisive role in the DNA chain [1].However, the availability of P is limited for most natural ecosystems and plant growth [2].P is particularly important in Chilean volcanic soils with high P retention or fixation [3].In general, when synthetic phosphorus fertilizers are applied to this type of soils, between 5 and 25% of the applied P is absorbed by plants, while the remaining (75-95%) is fixed or retained in insoluble forms by soil colloids, active complexes of Al, Fe, Ca and organic matter [4], and then used by subsequent crops.By thus, new fertilizer applications are required to maintain high production rates.Chilean volcanic soils present high contents of total P and organic matter, and high acidity.These type of soils, classified as Andisols, are the least extensive soil order [5] and occupy only 0.7% of the earth's land surface or just below 963,000 km 2 .Nevertheless, in Chile, volcanic soils support the bulk of agricultural and forestry production, covering more than 5.3 × 10 6 hectares and representing nearly 50-60% of the country's arable land [3].Wheat (Triticum aestivum L.) is one of the most important crops in Chile and it is mainly produced by small farmers [6].Most of Chilean wheat production is obtained from volcanic soils, while wheat productivity is based on the use of varieties with high yield potential, which requires the intensive use of inputs, such as fertilizers, calcareous amendments, and pesticides [7].The high P uptake rate in the early growth stages of wheat demands a high P amount from soil [8].P uptake by wheat starts from the first stage of the plant vegetative development (establishment) of the plant, and continues through the reproductive development until the maturity stage of the crop (anthesis and grain filling).This occurs because P is transported inside the plant passing from old to young tissues, and finally, the grain becomes the final supply of P [9].However, fertilizers applications can have a negative effect on the environment if they are not properly managed, i.e., increasing losses by leaching, runoff of nutrients, especially nitrogen (N) and P. Some of the reasons for these problems are low use efficiency of fertilizers and the continuous long-term use [10].
In this regard, soil microorganisms, such as bacteria, have a potential role in developing sustainable systems for plant growth [11].The use of biofertilizers and microbial biostimulators is considered an environmentally acceptable practice to reduce external inputs and improve the quantity and quality of internal resources [12].In fact, microorganisms, such as phosphate solubilizing bacteria (PSB), can provide crops with available P from barely soluble forms in the soil and physiologically active substances that trigger a higher metabolic activity once they interact with the plant [10].Therefore, the inoculation of Bacillus thuringiesis improved the solubilization of sparingly soluble phosphate compounds in soils, resulting in a higher crop yield and increase the concentration of soluble P in soil, and the supply of this nutrient in plants [13].On the other hand, continuous applications of P at rates exceeding plant uptake result in an accumulation of insoluble P in the soil [10].
Among the different strategies adopted by microorganisms to solubilize P, the involvement of low-molecular mass organic acids (OA) secreted by microorganisms has been a well-recognized and widely accepted theory as a primary means of P solubilization, and several studies have identified and quantified organic acids and define their role in the solubilization process [14,15].Acidification of the microbial cells and their surroundings allows for the release of P ions from the mineral P by replacing H + by Ca 2+ .The efficiency of solubilization, however, depends on the kind of organic acids released into the medium and their concentration.Furthermore, the quality of the acid is more important for P-solubilization than the total amount of acids produced by PSB [16].Simultaneous production of different OAs by PSB strains may contribute to a greater solubilization potential of insoluble inorganic phosphates [15].Additionally, some inorganic acids, such as hydrochloric, nitric, and sulfuric acids, produced by chemoautotrophs and the H + pump, contribute to soil P solubilization.Inorganic acids released as tricalcium phosphate by converting to di-and monobasic phosphates result of an enhanced availability of the P in the soil and to the plants [1].According to Freitas et al. [17], B. thruringiensis represented the rhizobacteria category of P-solubilizers that acidified the pH of medium.In addition, the Bacillus have an advantage over other bacteria due to their ability to form endospores, and thus resist changes in environmental conditions leading to high stability as biofungicides or biofertilizers [18].B. thruringiensis has been isolated from the wheat rhizosphere and some of the strains had the ability to solubilize phosphates at rates in the range of 15.05 to 16.65 µg mL −1 , depending of the isolated strain the most successful ones were isolated from arid and alkaline soils [19].However, to our knowledge, these strains have not been tested in wheat crops and soil with low P availability.
Knowing the problem of the P in volcanic soil, the application of PSB, such as B. thuringiensis, would contribute to increase the availability of this nutrient for plants, and reduce the need for the application of synthetic P fertilizers, and the environmental effect associated with excess of P applications.The objective this study was to assess the availability of P in soils that were derived from volcanic ash in two sites with soils of different taxonomic order (Andisol and Ultisol), inoculated with P solubilizing bacteria (B.thuringiensis), and their concentration in wheat plants at different growth stages.

Experimental Site
The experiment was conducted in pots under greenhouse conditions at El Nogal Experimental Station of the Faculty of Agronomy, University of Concepción, Chillán, Bio Bio Region, Chile (36 • 35 43.2 S, 72 • 04 39 W, and 140 ma.s.l).

Site I
It corresponded to the order Andisol of the Santa Barbara series (Typic Haploxerands) [20] whose samples were obtained in El Carmen (19

Experimental Design
The experimental design applied was completely randomized and consisted of four treatments, realizing destructive sampling four replicates for each treatment in three different times according Zadoks scale (Z46, Z66 and Z87).The following treatments were applied in both soils (Andisol and Ultisol): C = control; S + Bt = soil inoculated with B. thuringiensis; SE = soil sterilized; and, SE + Bt = soil sterilized and inoculated with B. thuringiensis.
All of the management treatments received a corrective fertilization for macro and micronutrient deficiency, according to the nutrient demand for wheat and the soil analysis for an expected yield of 8 t ha −1 .Excluding the applications of P. Site II (Ultisol) received an application of 2 t ha −1 of limestone to correct soil acidity and 0.5 t ha −1 of calcium sulfate to correct calcium and sulfur limitations in both soils.
Sterilization of soil in the respective treatments (SE and SE + Bt) was conducted using an autoclave at 121 • C for 1 h for two times with two days interval (soil without intervention) between each sterilization [24].In addition, destructive sampling of the pots was carried out for both soil and plant analyzes at 46, 66, and 87 days after sowing (DAS), according to the development scale proposed by Zadoks: Stem elongation (Z46), Anthesis (Z66), and Dough development stage (Z87) [25].

Pots
Pots with a capacity of 3.5 kg were filled with 2.5 kg of soil and seven wheat seeds (Triticum aestivum L.) var.Pantera INIA variety were sown.The seeds were not sterilized and they were provided as certified seeds by the National Institute for Agriculture and Livestock Research (INIA).After plant emergence, four plants were inoculated with strain of B. thuringiensis in a concentration of 10 6 CFU mL −1 (colony forming unit).The experiment was carried out under greenhouse conditions.Soil moisture in the pots was maintained at 70% of field capacity by using a moisture sensor (TDR) and distilled water.Irrigation was applied manually.

Origin of the Strain and Inoculum Preparation
AG-82 strain B. thuringiensis was used.These were obtained from the microbial collection of the Faculty of Agronomy, University of Concepción, Chillán, Chile.These were isolated from the rhizosphere of industrial chicory plants (Cichorium intybus L.), capable of solubilizing 117.32 mg L −1 P in vitro [26].Strains were multiplied using the methodology, as described by Slack and Wheldon [27], which, consisted in reproducing in standard nutrient broth I (Merck) under constant agitation at 150 rpm and 25 • C for two days (Lab Companion model SI-600 benchtop shaker; Lab Companion, Jeio Tech's laboratory instrument brand, Daejeon, South Korea).In order to determine the concentration, the optical density was measured in a spectrophotometer (MECASYS, POP Optizen Bio, Daejeon, South Korea) at 600 nm and its equivalence in CFU mL −1 was calculated.

Inoculation
Plants were inoculated at 20 DAS with PSB (B.thuringiensis) at a concentration of 10 6 CFU mL −1 .For inoculation, the bacteria were suspended in sucrose at 1%, or 10 g L −1 w/v [26]; and then, applied to the soil by adding 5 mL around each plant.For the treatments with no use of PSB (control), the 1% sucrose solution (previously submitted to autoclave treatment) was applied at the same rate (5 mL) around each plant.The use of sucrose in both cases is to avoid osmotic shock of the cells if pure water is used.At 46 DAS, a re-inoculation was performed to strengthen and ensure the presence of bacteria in the soil and sucrose (previously submitted to autoclave treatment) in the control treatments (C and SE).

Analysis of P in the Plants and Soil and pH Levels
The P in the tissues was determined by drying the samples at 65 • C, and then grinding them to a total passage through a 1.0 mm sieve, then 1.0 g of the samples were weighed and subsequently calcined at 500 • C for 5 h, and the ashes were dissolved in HCl at 2 mol L −1 , then heated at 120 • C for 40 min.They were then filtered and the P determined by colorimetry with nitro-vanado-molybdate at 720 nm [27].Olsen-P was extracted with Na 2 CO 3 0.5 mol L −1 , pH 8.5, and also determined by colorimetric method at 820 nm.Measurements of the pH in H 2 O suspension and potentiometric determinations (soil:water ratio of 1:2.5) were conducted according to the methods recommended for Chilean soils [21,28].

Enzymatic Activity and Soil Microbial Biomass
All of the samples (those taken at Z46, Z66 and Z87 growth stages) were stored in a cold room at 4 • C prior to measurements of enzymatic activity and soil microbial biomass.The samples were stored prior to measurements for five days.
Acid phosphatase activity in soil was determined using as substrate: P-nitrophenyl disodium phosphate (PNPP 0.115 M), 2 mL of 0.5 M sodium acetate buffer at pH 6.5, using acetic acid, and a volume of 0.5 mL of substrate of 0.5 g of sieved soil (<2 mm), and then incubated at 37 • C for 30 min [29], and absorbance was read in a spectrophotometer against a reagent blank.
Microbial soil biomass activity was determined by hydrolysis of fluorescein diacetate (FDA).For which, 1.0 g of wet soil was weighed in screw cap test tubes (samples were in triplicate and a blank was also included), then 9.9 mL of sodium phosphate buffer and 0.1 mL of FDA were added with subsequent stirring and brought to the thermoregulated bath at 25 • C for 1 h, then samples were withdrawn and placed in an ice bath.10 mL of acetone was added, shaken and filtered, and absorbance was read at 490 nm in a spectrophotometer against a reagent blank [30].

Plant Yield
Yield was evaluated by cutting aerial parts of the wheat plants tissue at Z46, Z66, and Z87 growth stages.Tissue samples were cut and separated from the root and washed with abundant P-free distilled water.Samples were stored in paper bags and weighed fresh in a high precision scale (Mettler-Toledo ® , BB2440, Greifensee, Switzerland).Then, dried in a forced-air drying oven (Memmert ® , 854 Schwabach, Germany) at 65 • C for 72 h to constant weight.

Statistical Analysis
The results were tested for normality (Shapiro-Wilks), homogeneity of variances and one-way analysis of variance (ANOVA) was used to determine significant treatment effects at 5% significance level.The Tukey test (p < 0.05) was applied for the comparison of means using the statistical package SPSS Version 23.0 for MAC OS [31].

Results
The effect of the inoculation with B. thuringiensis in the two sites under study (Andisol and Ultisol) was expressed as a positive or negative effect on the chemical and microbiological properties of the rhizosphere, plant growth, and P nutrition in the inoculated rhizosphere.Corresponding effects observed in the uninoculated rhizosphere are attributed to the plant itself.

Soil Chemical Properties
B. thuringiensis inoculation resulted in no significant differences in pH in all of the treatments when compared to the controls during Stem elongation (Z46), according to Zadoks growth scale.The same occurred in the Anthesis phase (Z66) and in the Dough development (Z87) grow stages (Table 1).The Olsen-P remained similar between treatments and variable at different sampling time (Table 1).The concentration of soil P did not increase significantly with the inoculation in all of the treatments for the Z46, Z66, and Z87 growth stages (Table 1).These are generally low values according to the reference values for the interpretation of the soil analysis methods recommended for Chilean soils [21].
3.1.2.P in the Plant P concentration in plants (aerial tissues) showed a significant increase of 34% in the SE + Bt treatment at Z46 growth stage when compared to the SE, and no significant differences were found between the inoculated soil and its control (C) (Figure 1).The same occurred at stage Z66 as inoculation resulted in a 21% increase of P concentration in the S + Bt treatment when compared to the C.However, the SE + Bt treatment decreased by 46% compared to the SE (Figure 1).In addition, P concentration in plants decreased 13% in S + Bt at stage Z87 as compared to the C.No significant differences were observed between SE + Bt and the SE (Figure 1).The Olsen-P remained similar between treatments and variable at different sampling time (Table 1).The concentration of soil P did not increase significantly with the inoculation in all of the treatments for the Z46, Z66, and Z87 growth stages (Table 1).These are generally low values according to the reference values for the interpretation of the soil analysis methods recommended for Chilean soils [21].
3.1.2.P in the Plant P concentration in plants (aerial tissues) showed a significant increase of 34% in the SE + Bt treatment at Z46 growth stage when compared to the SE, and no significant differences were found between the inoculated soil and its control (C) (Figure 1).The same occurred at stage Z66 as inoculation resulted in a 21% increase of P concentration in the S + Bt treatment when compared to the C.However, the SE + Bt treatment decreased by 46% compared to the SE (Figure 1).In addition, P concentration in plants decreased 13% in S + Bt at stage Z87 as compared to the C.No significant differences were observed between SE + Bt and the SE (Figure 1).The inoculation significantly decreased the P concentration in plant (radical tissue) at Z87. S + Bt and SE + Bt recorded decreased in 75% and 34% when compared to the C and SE, respectively (Figure 2).The inoculation significantly decreased the P concentration in plant (radical tissue) at Z87. S + Bt and SE + Bt recorded decreased in 75% and 34% when compared to the C and SE, respectively (Figure 2).The Olsen-P remained similar between treatments and variable at different sampling time (Table 1).The concentration of soil P did not increase significantly with the inoculation in all of the treatments for the Z46, Z66, and Z87 growth stages (Table 1).These are generally low values according to the reference values for the interpretation of the soil analysis methods recommended for Chilean soils [21].

P in the Plant
P concentration in plants (aerial tissues) showed a significant increase of 34% in the SE + Bt treatment at Z46 growth stage when compared to the SE, and no significant differences were found between the inoculated soil and its control (C) (Figure 1).The same occurred at stage Z66 as inoculation resulted in a 21% increase of P concentration in the S + Bt treatment when compared to the C.However, the SE + Bt treatment decreased by 46% compared to the SE (Figure 1).In addition, P concentration in plants decreased 13% in S + Bt at stage Z87 as compared to the C.No significant differences were observed between SE + Bt and the SE (Figure 1).The inoculation significantly decreased the P concentration in plant (radical tissue) at Z87. S + Bt and SE + Bt recorded decreased in 75% and 34% when compared to the C and SE, respectively (Figure 2).

Soil Biological Properties
Acid phosphatase activity decreased 42 and 60% with the inoculation in S + Bt and SE + Bt treatments at Z46 stage when compared to the C and SE (Table 1).A significant increase of 62 and 100% was also observed in treatments S + Bt and SE + Bt at Z66 stage (Table 1), while values increased 70 and 60% with the inoculation in treatments S + Bt and SE + Bt at Z87 stage as compared to the C and SE (Table 1).Regarding microbial biomass of the soil, the inoculation significantly increase of 88 and 218% in S + Bt and SE + Bt at Z46 growth stage compared to the C and SE (Table 1).At the Z66 growth stage, it decreased 52% in S + Bt and 60% in SE + Bt when compared to the C and SE, respectively (Table 1).At the Z87 growth stage, microbial biomass increased significantly in S + Bt (114%) when compared to the C, while it decreased by 31% in SE + Bt compared to the SE, respectively (Table 1).

Plant Biomass
Aerial biomass of the plants decreased significantly with the inoculation at all stages under study (Z46, Z66, and Z87) in S + Bt when compared to the C.However, no significant differences were found between SE + Bt and the SE, respectively (Table 2).The inoculation did not increase the root biomass at Z46 and Z66 growth stages, showing no significant differences (Table 3).For the Z87 growth stage, root biomass decreased significantly in SE + Bt when compared to the SE (Table 2).Values of pH did not show significant differences with the inoculation at Z46 and Z86 growth stages.However, a significant increase of 0.33 units was observed in S + Bt at Z66 stage when compared to the C (Table 3).
Olsen-P increased 11% in SE + Bt when compared to the SE, but it decreased 14% in S + Bt at Z46 stage compared to the C (Table 3).A significant decrease of 18% was also observed in SE + Bt at Z66 growth stage compared to the SE (Table 3).At Z87 stage, no significant differences were observed with the inoculation between S + Bt and the C or SE + Bt and the SE (Table 3).For example, these values are in general means according to the reference values for the interpretation of the soil analysis methods recommended for Chilean soils [17].

P in the Plant
The P concentration in plants (aerial tissue) increased significantly by 20 and 67% with inoculation treatments S + Bt and SE + Bt, respectively, at Z46 stage compared to the C and SE.However, for the Z66 P in the plant significantly decreased 25% in SE + Bt treatment compared to the SE, showing no differences in the unsterilized treatments.Instead, the P concentration in plants increased significantly 22% in the S + Bt treatment compared to the C in stage Z87 (Figure 3).Values of pH did not show significant differences with the inoculation at Z46 and Z86 growth stages.However, a significant increase of 0.33 units was observed in S + Bt at Z66 stage when compared to the C (Table 3).
Olsen-P increased 11% in SE + Bt when compared to the SE, but it decreased 14% in S + Bt at Z46 stage compared to the C (Table 3).A significant decrease of 18% was also observed in SE + Bt at Z66 growth stage compared to the SE (Table 3).At Z87 stage, no significant differences were observed with the inoculation between S + Bt and the C or SE + Bt and the SE.(Table 3).For example, these values are in general means according to the reference values for the interpretation of the soil analysis methods recommended for Chilean soils [17].

P in the Plant
The P concentration in plants (aerial tissue) increased significantly by 20 and 67% with inoculation treatments S + Bt and SE + Bt, respectively, at Z46 stage compared to the C and SE.However, for the Z66 P in the plant significantly decreased 25% in SE + Bt treatment compared to the SE, showing no differences in the unsterilized treatments.Instead, the P concentration in plants increased significantly 22% in the S + Bt treatment compared to the C in stage Z87.(Figure 3).The inoculation increased 26% the P concentration in roots in treatment S + Bt when compared to the C Z87 days after sowing.No differences were found in the rest of the treatments (Figure 4).The inoculation increased 26% the P concentration in roots in treatment S + Bt when compared to the C Z87 days after sowing.No differences were found in the rest of the treatments (Figure 4).

Soil Biological Properties
The activity of the acid phosphatase enzyme did not show significant differences with the inoculation at Z46 and Z66 stages, (Table 3).For Z87 stage, the S + Bt treatment had a significant decrease of 16% compared to the C (Table 3).On the other hand, the inoculation decreased the microbial biomass 42% in the SE + Bt treatment at Z46 stage compared to the SE.No significant differences were found in the other treatments and microbial biomass significantly increased 947 and 502% in treatments S + Bt and SE + Bt compared to the C and SE at Z66 stage.For Z87 stage, the SE + Bt treatment increased 72% compared to the SE (Table 3).

Plant Biomass
Aerial biomass did not show significant differences between the inoculated treatments and the controls.However, at Z87 stage, aerial biomass decreased 30% in SE + Bt compared to the SE (Table 4).

Soil Biological Properties
The activity of the acid phosphatase enzyme did not show significant differences with the inoculation at Z46 and Z66 stages, (Table 3).For Z87 stage, the S + Bt treatment had a significant decrease of 16% compared to the C (Table 3).On the other hand, the inoculation decreased the microbial biomass 42% in the SE + Bt treatment at Z46 stage compared to the SE.No significant differences were found in the other treatments and microbial biomass significantly increased 947 and 502% in treatments S + Bt and SE + Bt compared to the C and SE at Z66 stage.For Z87 stage, the SE + Bt treatment increased 72% compared to the SE (Table 3).

Plant Biomass
Aerial biomass did not show significant differences between the inoculated treatments and the controls.However, at Z87 stage, aerial biomass decreased 30% in SE + Bt compared to the SE (Table 4).Root biomass increased 40% in S + Bt and SE + Bt in 23% at stage Z46 when compared to the C and SE, respectively (Table 4).At stage Z66, (Table 4).Finally, at stage Z87, root biomass increased 17% in S + Bt as compared to the C (Table 4).

Discussion
The present study used the B. thuringiensis that was isolated in the rhizosphere of industrial chicory plants (Cichorium intybus L.) [26], and then inoculated in wheat plants growing in two different soils (Andisol and Ultisol).Due to fact that our methodological approach includes soil sterilization and the known effects of soil sterilization with respect to chemical, microbiological and physical change; for this reason, in this study we only focused on comparing the effects of microorganisms between treatments C vs. S + Bt and SE vs. SE + Bt (control or natural soil without inoculation and inoculated; and, similarly, for sterilized soil).In this study was not so relevant to compare the effects of the sterilization of the soil, but of the inoculation; that is why we had a control for each approach.Otherwise, a soil characterization after autoclaving was needed.Therefore, considering these issues, the results showed that the inoculation was not positive in Andisol in case of pH, available P and biomass productivity (Tables 1 and 2), but had a positive effect in P concentration in aerial tissue (Figure 1), microbial biomass and phosphatase activity (Table 1).In case of Ultisol, the inoculation increases pH 0.3 units, available P in 11% (Table 3), root biomass (Table 4), P concentration in tissue (Figures 3 and 4) and microbial biomass (Table 3).Soil microorganisms play a vital role in P acquisition and transformation in soil, and thus, affect the subsequent availability of P to the plant roots, but the positive or negative effects these organisms depend on much factors.The negative effect of soluble P on microbial acid productivity might also be responsible for final soluble P concentration.Also, the decrease in the concentration of organic acid produced by the bacteria would be expected due to (1) typical short life cycle (32 to 48 h) of Bacillus spp.; (2) population growth in constrained environment may decline after 28 to 90 h of incubation [32].Furthermore, organic acid may precipitate and chelate with cations in the soil [14].All of these may contribute to the limitations of biofertilizer application of PSB.
The pH increased with the inoculation at stage Z66 (Ultisol), whose results agree with those that are indicated by Dinesh et al. [33], while Chen et al. [34] reported that the application of PSB reduced soil pH under certain conditions due to the release of organic acids to solubilize the insoluble P in the soil layers.Other microbial metabolites are also responsible for reducing soil pH.This may be a strategy to increase the availability of P or other nutrients in the soil and provide a greater uptake for plants through changes in the pH rhizosphere induced by roots or microorganisms.The increase in available P, by 11%, with inoculation (Z46 Ultisol) has also been reported in previous studies [34,35].In turn, Ogut and Er [35] also observed an increase in the available P in the rhizosphere of wheat inoculated with Bacillus sp. after 31 days of the Zadoks scale.Furthermore, Ul Hussan and Bano [36] also reported an increase of available P in the rhizosphere of wheat plants inoculated with Bacillus cereaus.These findings, shown that the increase of P in the rhizosphere can provide a better development of the plants.The non-significant increase of Olsen-P at Z46, Z66 and Z87 stages (in Andisol) with inoculation, may be due to P immobilization by the microorganisms and P fixation, retention by the soil colloids or others factors, as mentioned earlier, which affect P solubilization by microorganisms in soil.
The significant increase in P concentration in aerial tissue (Andisol and Ultisol), and in root tissue (Ultisol) in wheat plants with inoculation was also observed by Schoebitz et al. [37], who highlights the important role that PSB play in plant nutrition by increasing available P in soil.A study conducted by Morales et al. [38] in an Ultisol soil showed that inoculation with Penicillum albidum significantly affects P in both plant and roots.In addition, Turan et al. [39] reported increases in P in plant tissues inoculated with Bacillus magaterium.The multidimensional functions of PGPRs also contribute to a greater uptake of nutrients by plants, leading to a higher content of P and other elements in the upper parts of plants.Ogut and Er [35] found evidence of the increase of P in plant tissues of inoculated for Bacillus and others strains in wheat after 31, 61, and 92 days, according to Zadoks growth scale.
The reduction of P in the aerial and root tissues at Z66 (especially in the case of sterilized treatment, Andisol) Z87 stages may be attributed to the low availability of P in the soil caused by P immobilization by microorganisms, and other soil reactions that are involved in P availability.The microbiologically solubilized P in the rhizosphere can be immobilized by soil microorganisms or by physicochemical reactions of the soil.In addition, as the roots are responsible for the absorption of essential nutrients, the low concentration of P in plants grown in the sterilized treatments may be caused or influenced by the poor root development that was observed in these treatments (root biomass; Andisol).
The increase in acid phosphatase activity observed at later growth stages in this study (Andisol) and decrease in Ultisol Z87, which may be attributed to the P forms in soil.The relationship between P-solubilization and acid production is understandable, whereas the apparent relationship with phosphatase production is misleading.Phosphatases catalyze the hydrolysis of organic P-compounds, such as esters and anhydrides of orthophosphoric acid, and do not solubilize rock phosphates [17].This enzyme is important in soil, in particular for the Andisols, where the P is limiting for vegetable development, and our results agrees with those obtained by other authors in previous studies [38,39].This can be attributed to the increase in microbial biomass activity in the inoculated soil.These enzymes, which are released outside the cell (exo-enzymes), are non-specific in nature and use organic P as a substrate to convert it into an inorganic form.This enzyme is crucial for the mobilizing P in soils with a high content of organic P [40].The increase in the microbial biomass of inoculated soil (Andisol and Ultisol) has also been described in the literature; e.g., Dinesh et al. [33] reported an increase in microbial biomass in soil that was inoculated with plant growth promoting Rhizobacteria (PGPR).In addition, a study conducted by Wang et al. [13] showed that the PSB population increased in the soil with the inoculation in the first 60 days, followed by a significant decrease over time.Metabolic activities are responsible of a number of processes in the soil, such as organic matter mineralization and humification, which in turn, affect other processes involving fundamental elements in the soil (C, N, P, and S), as well as all of the transformations involving the microbial biomass of the soil [41].These microorganisms can mobilize P from unavailable forms so that an increase of the microbial biomass can promote a greater availability of P and other nutrients to plants.All of the processes and functions occurring in the rhizosphere are controlled by the activities of plant roots, rhizosphere microorganisms and interactions between roots and microorganisms [42].Specific characteristics of our soils; e.g., high particle surface area (Andisol), which allows for fluorescein diacetate immobilization within the soil environment, which could come from many microbial and plant species, could interfere in the FDA determination, and eventually explain early biomass in the experiment (e.g., Table 2; SE, SE + Bt).On the other hand, Fe or Al content (Ultisol and Andisol), could also interfere in the determination making difficult to achieve consistent results.On the other hand, microorganisms that for surviving steam sterilization produce spores could after 30-40 days starts their activity.
Inoculation did not affect significantly the aerial biomass of the plants at all of the growth stages evaluated (Andisol and Ultisol), could be related to adequate soil available P dynamics in our greenhouse conditions, or which could hint at a negative interaction between the inoculum and the resident soil microorganisms.Wani et al. [41] observed that inoculation with Bacillus or Pseudomonas alone did not significantly affect the biomass of chickpea plants 45 and 90 days after sowing.Ogut and Er ([35], observed increases in those parameters in wheat plants inoculated with Bacillus sp. and other strains.The increase of root biomass due to inoculation (at earlier stage, Z46; and Z87, Ultisol) in this study, has been reported in several studies [13].Different growth promoting characteristics of the PGPR and PSB bacteria are related to a greater growth of the roots of plants [43].The growth of the roots in response to inoculation of bacteria also leads to a better absorption of nutrients by plants, particularly P, and other functions in the rhizosphere of plants.The low development that was observed in aerial and root biomass (Andisol) caused for the inoculation is one the negative effect observed in this study, and affected significantly the plant growth, especially in the sterilized treatments.Alphei and Scheu [44], determined that the sterilization using autoclaving and gamma irradiation caused least side effects in C, N and P-status of the soil.These differences are due to the effect of sterilization treatments on the physicochemical and biological properties of the soil.In fact, soil sterilization usually results in reduced plant growth due to the toxic effect of Mn available in the soil released by the organic fraction and the elimination of microorganisms [45].In addition, sterilization in some soils affects plant growth due to the elimination of microorganisms responsible for the mineralization processes of nutrients such as N, P, and S. Mahmood et al. [46] observed a significant increase in the macronutrients (NH 4 + , NO 3 − , P, and pH of the soil) of wheat plants as effect to autoclave sterilization.These positive or negative effects depend on the type of soil.In addition, the effects of sterilization on soil properties mainly depend on the technique used and the time of exposure of the soil.The contrasting effects caused by inoculation with B. thuringiensis in Andisol in relation Ultisol and the growing plants were as expected.Differences are mainly attributed to the physical, chemical, biological characteristics of soils, and soil management practices.Andisols have particular soil characteristics: e.g., low bulk density, high content of organic C, high P retention.P sorption to soil minerals (like allophane, imogolite, and Fe-or Al-oxides associated with humic substances); and, Al present in the interlayers of expandable phyllosilicates in these soils [47,48], can protect them against microbial and enzymatic decomposition [49] and can difficult their response to inoculation.In general, the inoculation had both positive and negative effects on the P uptake by plants and on the availability of P in the soil (Olsen-P).However, for a definite use of PSB in the field, the experiments we have carried out should ideally be repeated with a different matrix close to that probably being applied in the field According to Jorquera et al. [50], the bioavailability of P depends on the solubility and structure of its chemical forms in the soil-root environment, as well as on the susceptibility to microbial attack.The use of phosphobacteria as inoculants in agricultural/grazing systems in volcanic soils can help to reduce applications of P fertilizer, resulting in lower environmental pollution and promoting sustainable agriculture in Chile [50].

Conclusions
Our study demonstrates effect on the use of P-solubilizing B. thuringiensis as inoculate in two soil types of volcanic origin.Results in the Andisol show a negative effect in important parameters in the wheat plants and soil, but in the Ultisol results, show positive effects for most of the parameters measured at the different times.Further research is needed especially for the former, given its particular soil characteristics.

Figure 1 .
Figure 1.Effects of B. thuringiensis inoculation on the P concentration in wheat plants (aerial part) at different growth stages of the Zadoks scale.(Andisol).Means values with the same letter are not significantly different according to Tukey test (p > 0.05).(C = control; S + Bt = soil inoculated with B. thuringiensis; SE = soil sterilized; SE + Bt = soil sterilized and inoculated with B. thuringiensis; Z = days after sowing according to the Zadoks scale).

Figure 2 .Figure 1 .
Figure 2. Effects of B. thuringiensis inoculation on P concentration in roots of wheat plants in the Z87 (Andisol).Mean values with the same letter are not significantly different according to Tukey test (p Sustainability 2018, 10, x FOR PEER REVIEW 6 of 15

Figure 1 .
Figure 1.Effects of B. thuringiensis inoculation on the P concentration in wheat plants (aerial part) at different growth stages of the Zadoks scale.(Andisol).Means values with the same letter are not significantly different according to Tukey test (p > 0.05).(C = control; S + Bt = soil inoculated with B. thuringiensis; SE = soil sterilized; SE + Bt = soil sterilized and inoculated with B. thuringiensis; Z = days after sowing according to the Zadoks scale).

Figure 2 .Figure 2 .
Figure 2. Effects of B. thuringiensis inoculation on P concentration in roots of wheat plants in the Z87 (Andisol).Mean values with the same letter are not significantly different according to Tukey test (p ± 0.02 c 0.01 ± 0.01 c Mean values with the same letter in the column and Zadoks scale are not significantly different according to Tukey test (p > 0.05).(C = control; S + Bt = soil inoculated with B. thuringiensis; SE = soil sterilized; SE + Bt = soil sterilized and inoculated with B. thuringiensis; Z = Zadoks scale in days after sowing; ± = standard error).

Figure 3 .Figure 3 .
Figure 3. Effects of B. thuringiensis inoculation on the P concentration in wheat plants (aerial part) at different growth stages of the Zadoks scale (Ultisol).Mean values with the same are not significantly different according to Tukey test (p > 0.05).(C = control; S + Bt = soil inoculated with B. thuringiensis;

Sustainability 2018 ,
10, x FOR PEER REVIEW 9 of 15 SE = soil sterilized; SE + Bt = soil sterilized and inoculated with B. thuringiensis; Z = days after sowing according to the Zadoks scale).

Figure 4 .
Figure 4. Effects of B. thuringiensis inoculation on P concentration in roots wheat plants in the Z87 (Ultisol).Mean values with the same letter are not significantly different according to Tukey test (p > 0.05).(C = control; S + Bt = Soil inoculated with B. thuringiensis; SE = soil sterilized; SE + Bt = soil sterilized and inoculation with B. thuringiensis; Z = days after sowing according to the Zadoks scale).
± 0.23 b 0.43 ± 0.01 b 2.42 ± 0.09 a 0.48 ± 0.01 b 2.54 ± 0.19 b 0.06 ± 0.0 d Means with the same letter in the column and Zadoks growth scale are not significantly different according to Tukey test (p > 0.05).(C = control; S + Bt = soil inoculated with B. thuringiensis; SE = soil sterilized; SE + BT = soil sterilized and inoculated with B. thuringiensis; Z = Zadoks scale in days after sowing; ± = standard error).

Figure 4 .
Figure 4. Effects of B. thuringiensis inoculation on P concentration in roots wheat plants in the Z87 (Ultisol).Mean values with the same letter are not significantly different according to Tukey test (p > 0.05).(C = control; S + Bt = Soil inoculated with B. thuringiensis; SE = soil sterilized; SE + Bt = soil sterilized and inoculation with B. thuringiensis; Z = days after sowing according to the Zadoks scale).

Table 1 .
Effect of B. thuringiensis inoculation on pH, Olsen-P, acid phosphatase and soil microbial biomass in wheat plants at different growth stages according to Zadoks scale (Andisol).

Table 2 .
Aerial and root biomass influenced by inoculation with B. thuringiensis in wheat plants at different growth stages according to Zadoks scale (Andisol).

Table 3 .
Effects of B. thuringiensis inoculation on pH, Olsen-P, phosphatase and soil microbial biomass in wheat plants at different growth stages according to Zadoks scale (Ultisol).
Mean values with the same letter in the column and Zadoks scale are not significantly different according to Tukey test (p > 0.05).(C= control; S + Bt = soil inoculated with B thuringiensis; SE = soil sterilized; SE + Bt = soil sterilized and inoculated with B. thuringiensis; pNF = P-nitrophenyl; F = fluorescein; Z = Zadoks scale in days after sowing; ± standard error).
3.2.Effect of the B. thuringiensis Inoculation in an Ultisol in Different Growth Stages of Wheat Plants 3.2.1.Soil Chemical Properties

Table 4 .
Effect of B. thuringiensis inoculation on aerial and root biomass in wheat plants at different growth stages according to Zadoks scale (Ultisol).

Table 4 .
Effect of B. thuringiensis inoculation on aerial and root biomass in wheat plants at different growth stages according to Zadoks scale (Ultisol).
Means with the same letter in the column and Zadoks growth scale are not significantly different according to Tukey test (p > 0.05).(C = control; S + Bt = soil inoculated with B. thuringiensis; SE = soil sterilized; SE + BT = soil sterilized and inoculated with B. thuringiensis; Z = Zadoks scale in days after sowing; ± = standard error).