Introduction

Sustainable agricultural production relies on various factors such as environmental factors, soil quality, and nutrients bioavailability. Among nutrients, phosphorus (P) is the second most important element after nitrogen (N), which is crucial for completion of plant life cycle (Schröder et al. 2011; Cordell et al. 2011; Kochian 2012). The deficiency of P in soils may lead to leaf browning supplemented by smaller leaves, weaker main stem, and slower rate of development thus resulting in low crop yield (Vance et al. 2003; Rashid et al. 2005). Though a large amount of total P is present in most of the soils, merely a minor amount is instantly accessible to plants due to several factors such as soil pH, minerals, calcareousness, etc. According to an estimate, about 42% of the world’s cultivated lands are deficient in P. Since, a large proportion of soluble inorganic phosphate added to soil is rapidly fixed as insoluble forms soon after application and becomes unavailable to plants. Plants uptake P from soil as orthophosphate anions and in most of the soils; the concentration of orthophosphate in solution is low (typically 1–5 μM) (Schachtman et al. 1998; Porder and Ramachandran 2013).

Both the morphological and physiological features of roots are considered important in defining the capability for plants to facilitate the accessibility of crucial nutrients in soil medium (Darrah 1993; Rafique et al. 2019). Roots interact with soil microbes using a variety of mechanisms to get multiple benefits such as nutrients acquisition (Huang et al. 2014). The discovery of phosphate solubilizing bacteria dates back to almost century ago, and a noteworthy role of PSM on soil P dynamics and accessibility has been reported to date (Khan et al. 2009, 2010; Zhu et al. 2018). The PSB carry out 1–50% of P solubilization among all the microbes present in soil, while only 0.1–0.5% P solubilization is carried by phosphorus solubilizing fungi (PSF) (Tao et al. 2008). Consequently, use of PSB as biofertilizers for yield enhancement has gained attention of many researchers for many years especially belonging from genera, i.e., Azospirillum, Alcaligenes, Arthrobacter, Acinetobacter, Bacillus, Burkholderia, Pseudomonas, Enterobacter, Erwinia, Rhizobium, and Flavobacterium (Umair et al. 2018). Regarding P accessibility in rhizosphere, effective liberation of P by PSB from an immovable/adsorbed shape is an imperative feature. Microbes integrate soluble P thus preventing it from fixation/adsorption (Mohammadi 2012; Rafique et al. 2019).

Another important organic amendment that can enhance the fertility status of soil is biochar (BC), which is a carbon-rich amendment and may also contain appreciable amount of certain nutrients (Rajkovich et al. 2012; Azhar et al. 2019; Qayyum et al. 2019). Mostly, BC is recognized as a soil conditioner that has relatively very fine particles and capability to modify the soil properties (physical, chemical, and biological) (Rizwan et al. 2016; Zama et al. 2018). Since BC is highly resistant against the microbial decomposition, it is stable in soils for longer periods compared with the organic amendments (Qayyum et al. 2012; Rehman et al. 2017; Kern et al. 2019). Other benefits of BC include, improvement of water holding capacity, nutrients holding in soil, minimizing soil erosion, and promotion of microbial growth and activity in amended soil (Solaiman and Anawar 2015; Abbas et al. 2018). Moreover, the BC increases the P use efficiency of fertilizers through various mechanisms (Rafique et al. 2019; Ali et al. 2017; Glaser et al. 2002).

Previously, Wei et al. (2016) investigated the effect of BC and phosphorus solubilizing bacteria (PSB) on P fractions during composting process. Their findings show that combined application of BC and PSB increased the inorganic P fraction that was referred as plant available in the resulting composts. In another study, Rafique et al. (2017) reported positive effect of sawdust BC and PSB on maize plant height and nutrients uptake. Besides these, literature to date describes effect of PSB and BC independently on plant P nutrition but their interactive effect in alkaline calcareous soils where P deficiency is prevailing challenge is not studied. Thus, the present study was planned with objectives to investigate the interactive effect of PSB, BC, and different levels of fertilization on P bioavailability and maize (Zea mays L.) growth in an alkaline, calcareous, and poor soil. It was hypothesized that the P availability through PSB solubilization may be increased in BC-amended soil.

Materials and methods

Soil and biochar

It was a pot experiment following three factorial complete randomized designs. The experiment was conducted in a wire house of the Department of Soil Science, Bahauddin Zakariya University Multan (latitude 030°15′36″N; longitude 071°30′53″E). The soil used in the present experiment was fine silty, mixed, hyperthermic, Sodic Haplocambids (according to USDA classification). The detailed physicochemical properties of the soil are published elsewhere (Rehman et al. 2018). The BC used in the present study was prepared using rice straw as feedstock following Qayyum et al. (2015). The feedstock was collected from the paddy field from Multan. The dried and crushed rice straw was filled into vertical stainless steel silo-type reactor (30 cm diameter and 90 cm high). The material was combusted under anaerobic conditions for 4 hours after attaining 450 °C. The selected physicochemical properties of the BC are provided in Table 1.

Table 1 Selected physicochemical properties of the biochar
Full size table

Experimental setup

The experimental factors comprised the following: (i) BC (either no BC or BC at a rate of 2%, referred as B0 and B1), (ii) phosphorus solubilizing bacteria (no PSB and + PSB, referred as S0 and S1), and (iii) fertilizer addition in soil (no fertilizer (F0), half of required fertilizer (F1/2), full fertilizer (F1)). All treatments were replicated three times. The soil media of different treatments were prepared by mixing BC and respective fertilizer amendment and filled into clay pots of capacity 10 kg soil. The macro- and micronutrients except P were applied according to the recommended doses for maize in 10 kg of soil. The nutrients comprised 1.5 g N per 10 kg of soil using urea, 0, 0.3, or 0.6 g P using K2HPO4, 1.51 g K using K2H2PO4 and K2SO4, and 0.15 g Mg using MgSO4.7H2O. To avoid micronutrient deficiency, nutrient solution was applied in all pots following Qayyum et al. (2017). While for 2% BC treatment, the calculated amount of BC was mixed by hand in soil and then pots were filled.

The PSB solution (1% in 10% sugar solution) was prepared for inoculation of bacteria. The seeds of maize (variety 6119 hybrid Agroman) were dipped in the PSB solution for 2 hours and later dried on dry filter paper. Eight seeds were sown in each pot during end of monsoon season. After emergence of maize seedlings, three plants per pot were maintained. After the second week, only half dose of N fertilizer was added in respected treatments. The moisture content in the pots was maintained at 60% water holding capacity by calculating the weight loss at regular intervals and irrigating with ground water. After 45 days of growth, the morphological data of plants such as leaf area and plant height were measured, and plants were harvested. The shoot and roots were separated and packed separately for determination of fresh and dry masses.

The soil samples from pots as well as soil attached on root surfaces (termed as rhizosphere soil) were collected and processed for analyses.

Analyses

The characterization of BC was done following Mclaughlin (2010). The pH and EC of BC was determined in suspensions of BC and water at 1:5 ratio, respectively. The ash and volatile matter contents of BC were determined by combustion at 450 °C and 550 °C followed by calculation of weight loss. For P determination in BC, di-acid (nitric and perchloric acid in 2:1) digestion followed by color development and absorbance was measured by spectrophotometer at 420 nm (Murphy and Riley 1962).

The soil samples were analyzed for pH, EC, OM, total N, and Olsen’s P determination. Soil attached to roots was carefully separated and termed as rhizosphere soil, while the rest of the sample was referred as bulk soil. Both samples were separately analyzed for pH (1:5 suspensions of soil: water), EC (1:5 soil: water suspension), and Olsen’s P.

The plant samples were analyzed for total N, P, and K following recommended methodologies. For, N and P, 1.0 g oven dried, ground plant sample was digested (up to 350 °C) in 15 mL concentrated H2SO4 and 3.0 g digestion mixture (K2SO4 + CuSO4 @ 9:1). The total N concentration in digestate was measured following Kjeldahl method. The concentration of P in digestate was measured as described above for BC samples. For the K determination, the plant samples were digested in di-acid (perchloric and nitric acid mixture, 1:2) followed by determination on flame photometer.

Statistical analysis

Statistical analyses (three-way analysis of variance followed by Tukey HSD test) were done for all determined parameters using STATISTIX 8.1.

Results

Effect of PSB and BC on soil pH, EC, Olsen’s phosphorus, and total nitrogen

The interactive effects of BC, fertilizers, and PSB on soil pH and EC are given in Fig. 1. There was significant three-way interaction between investigated factors on soil pH and EC (Table 2). The BC application (B1) caused significant increase in soil pH at all fertilizer levels as compared with no-BC (B0). However, PSB application (S1) did not affect the pH values except in B0F0 where pH was decreased. Addition of BC also increased the soil EC as compared with B0 amendments. The maximum increase in soil EC was observed in B1S0F1 treatment.

Fig. 1
figure 1

Interactive effect of biochar (no biochar = B0 and biochar = B1), P fertilization (no fertilizer = F0, half of recommended = F1/2, full fertilizer = F1), and phosphate solubilizing bacteria (no PSB = S0, PSB = S1) on soil pH (a) and EC (b) of postharvest soil. Different letters presenting the bars are significantly different at P ≤ 0.05

Full size image
Table 2 Three-way analyses of variance table (F values) for investigated parameters
Full size table

The Olsen’s P was measured in rhizosphere soil samples (soil adjacent to the roots) as well as bulk soil sampled from the experimental pots (Fig. 2a, b). There was a significant three-way interaction for Olsen’s P in both parameters. Relatively higher values of Olsen’s P concentrations were observed in rhizosphere soil as compared with bulk soil. In the rhizosphere soil samples, higher P concentration was observed at B1S1F1 followed by B1S0F1 and B1S1F1/2. Both fertilizer treatments (F1/2 and F1) caused significant increase in Olsen’s P. Interestingly, B1S1F1/2 (half fertilizer application in combination with BC and PSB) caused similar values as B1S1F1 (full fertilizer application in combination with BC and PSB).

Fig. 2
figure 2

Interactive effect of biochar (no biochar = B0 and biochar = B1), P fertilization (no fertilizer = F0, half of recommended = F1/2, full fertilizer = F1), and phosphate solubilizing bacteria (no PSB = S0, PSB = S1) on rhizosphere Olsen’s P (a), bulk soil Olsen’s P (b), and soil total nitrogen (c) of post-harvest soil. Different letters presenting the bars are significantly different at P ≤ 0.05

Full size image

In bulk soil samples at full fertilizer and S1 (PSB), significantly higher values of Olsen’s P were found irrespective of BC application. However, B1F0S0 (no fertilizer, BC application in combination with no PSB), caused significantly higher Olsen’s P as compared with B0F0S0 (no fertilizer and no BC with no PSB). The soil total N concentration was higher in B0 (No BC) compared with B1 (BC application). The S1 (PSB) at all fertilizer treatments irrespective of BC decreased the total N except at F0B0 (no fertilizer and no BC) S1 (PSB) where significant increase in total N was found (Fig. 2c).

Effect of BC, PSB, and P fertilizer on plant growth attributes

The plant growth parameters are given in Fig. 3. The plants were taller in B0F1 (at both S0 and S1) and B1F1S0 treatments followed by B0F1/2. Results showed that increasing the P fertilizer rate significantly improved the plants’ height. However, except F1, BC caused lower values of plant height as compared with no BC treatment. Data regarding the fresh mass of plants have been reported in Fig. 3b. Statistical analysis revealed a significant (P ≤ 0.05) increase in the fresh mass of plants by increasing the fertilizer application rates. The inoculation of PSB also improved the fresh mass but only at F0 (no fertilizer) and B0F1/2 (no BC, and half fertilizer). The maximum fresh mass of plants was recorded in the B1F1 irrespective of PSB. The plant dry mass also followed same trend as the fresh mass in all treatments.

Fig. 3
figure 3

Interactive effect of biochar (no biochar = B0 and biochar = B1), P fertilization (no fertilizer = F0, half of recommended = F1/2, full fertilizer = F1), and phosphate solubilizing bacteria (no PSB = S0, PSB = S1) on plant height (a), fresh mass (b), and dry mass (c) of maize plants. Different letters presenting the bars are significantly different at P ≤ 0.05

Full size image

The concentration of N and P in plants

The results showed a significant interactive effect of BC, P fertilization, and PSB on N uptake by the maize plants (Fig. 4a). The application of PSB in the soil and control treatments did not affect N in plants. However, the addition of BC without PSB increased the N concentration in plants. The increase in plant N was observed where PSB and BC were applied in combination compared with other treatments. The highest plant N was noted in the B1S1F2, while the least plant N was noted in the control.

Fig. 4
figure 4

Interactive effect of biochar (no biochar = B0 and biochar = B1), P fertilization (no fertilizer = F0, half of recommended = F1/2, full fertilizer = F1), and phosphate solubilizing bacteria (no PSB = S0, PSB = S1) on nitrogen (a), and phosphorus (b) concentration in plants. Different letters presenting the bars are significantly different at P ≤ 0.05

Full size image

Correlation and principal component analysis

Based on correlation (Table 3) followed by the principal component analysis, the fresh mass (FM), dry mass (DM), bulk soil Olsen’s P, rhizosphere Olsen’s P, plant N, and plant P were comprehensively quantified to evaluate the optimal combined treatment of BC, fertilizer, and phosphate solubilizing bacteria. The comprehensive evaluation was done using the PASW 18.0 program. The correlation data showed significant values between plant growth parameters and soil P (both rhizosphere and bulk soil). The P concentration in plant was positively and significantly correlated with rhizosphere and bulk soil Olsen’s P. However, soil pH and EC did not correlate with agronomic as well as nutrients uptake by the plants. The correlation data was further analyzed for the principal component analysis (PCA). The Kaiser-Meyer-Olkin Measure of Sampling Adequacy value was neither very low nor high (0.624). However, the significant value of Bartlett’s Test of Sphericity was 0.000. Therefore, it was possible to continue for the PCA analysis. The initial eigenvalues (4.875, 2.215, and 1.011) showed three principal components which could influence by 81% on overall results of the study. Most of the investigated parameters (plant N, plant P, fresh mass, dry mass, plant height, and soil P) had higher values of component matrix showing higher dependency on component 1, while soil N and plant height had higher values of matrix for component 2, and soil EC with component 3. The same trend was visible on component plot at rotated space (Fig. 5). The residual regression values showed positive and higher values for the treatments B0F1/2S1, B0F1S1, B1F1/2S1, and B1F1S1 at component 1. The overall performance of B1F1S1 was ranked the highest compared with the rest of the treatments.

Table 3 Pearson correlation between investigated parameters
Full size table
Fig. 5
figure 5

Component plot in rotated space by performing principle component analysis of selected variables

Full size image

Discussion

The results of our study clearly demonstrated a significant influence of rice straw BC and PSB on the plant growth that was mainly due to improved soil properties and increased P availability. The BC used in our study increased the soil pH and EC at all fertilizer levels, which is attributed to high ash content and alkaline earth metals (Qayyum et al. 2015) and presence of OH functional groups (Liang et al. 2006; Jiang et al. 2012; Rizwan et al. 2016). In our study, the PSB decreased soil pH slightly in no BC treatments (Fig. 1). Similarly, slight decrease in soil pH and release of organic acids after application of PSB have been reported previously that may cause increase in P availability (He et al. 2002). Organic acids help to lower down the soil pH and promote solubility of inorganic P in soil. Soil EC was not affected significantly due to PSB, though it was previously reported that inoculation of PSB enhanced the EC of soil by releasing the alkaline nature phosphatase enzymes and by increasing the exchangeable cations (Jones 1998). The increase in soil EC with BC application is due to release of minerals especially K, Ca, and Mg (Glaser et al. 2002).

Our results clearly showed P solubilization by PSB that was enhanced in the presence of BC application that was due to decrease in the immobilization of P by making it mobile and bioavailable. The application of BC in soil enhances the availability of reduced carbon as a source of nutrition in soils. Moreover, the bacteria prefer highly porous materials for adsorption (Samonin and Elikova 2004) and BC best fits in this category. Increasing the population of PSB in rhizosphere may enhance their activities of P solubilization that was obvious from our results of Olsen’s P in the rhizosphere soil (Fig. 2). According to He et al. (2002), the PSB released the organic acids through their secretions in the soils, which dissolve the P minerals and make it soluble as well as labile. Simple effect of BC on P bioavailability is contradictory. In some studies, increased availability of P with BC has been reported (Steiner et al. 2007; Chan et al. 2008). However, our previous initial experiments showed reduction in availability of P with BC addition (un-published data) that might be due to high clay contents of soil (Bieleski 1973) and alkaline nature of BCs (Rehman et al. 2017; Abbas et al. 2018). However, in the present study, the combined effect of BC and PSB in the soil for P mobilization was the best as compared with the PSB and BC individual treatments (Fig. 2).

In our experiment, the addition of BC decreased the total N in post-harvest soil (Fig. 2). This may be due to depletion of N in those treatments where plant growth was better (Fig. 3). It is also confirmed through negative correlation between N and the rest of the parameters (Table 3). It is previously reported that BC, depending on feedstock, may contain small amount of N (Qayyum et al. 2012) that may attract the mycorrhizal fungi that significantly affects N availability in the plants (Topoliantz et al. 2005). The addition of BC along with N fertilizers may decrease the nitrification and ammonification processes which reduce the loss of N and increase its availability to plants (Lehmann 2006; Lehmann and Rondon 2006; Rondon et al. 2007). Yamato et al. (2006) reported that the improvement in the maize biomass in BC-applied soil was due to more efficient uptake of N.

The plant growth parameters also showed positive combined effect of BC, fertilizer, and PSB on growth parameters that was due to increased bioavailability of nutrients (K, N, and P). Results showed that increasing the amount of fertilizers increased plant height and biomass of maize plants. Meanwhile, the BC promoted plants height in half fertilizer treatment as well. The use of BC as amendment modifies the soil physicochemical properties and fertilizer use efficiency that ultimately influence plant growth which was obvious from increased plant height (Kimetu and Lehmann 2010; Rizwan et al. 2016). Rondon et al. (2007) argued that application of BC improves the microbial population in soil that helps in BNF as well as PSB that helps in solubilization of P (Alburquerque et al. 2015). Our data showed that in addition to P fertilizer, BC application enhanced the dry mass of plants. Similar to our results Blackwell et al. (2010) suggested that high application of BC along with full fertilizer doses improved the dry matter yield of crops. They justified positive effects as results of high sorption capacity of BC that ultimately reduces the N losses as well as immobilization of P.

Conclusion

The results showed that the combined application of rice straw BC and PSB at different levels of P fertilizers has significant influence on growth as well as nutrient uptake by the maize plants. The plant growth and nutrient uptake by plants increased with the applied amendments. The results demonstrated that the combined effects of amendments were higher than individual amendments. Thus, based on the results of the study, it is concluded that the combined use of PSB and BC in the P deficient and alkaline soils may increase the P uptake and yield of crops. The enhancement in the growth of the plants provides the information that interaction of BC with PSB could be a suitable approach for sustainable crop production. In the future, field investigations using varieties of BCs, PSB, and different crops are required for further understanding.