1. Introduction
Acid deposition has posed serious challenges to agricultural sustainability in China over the past few decades [
1]. Soil acidified slowly in natural conditions, which was mainly through the weathering of minerals and rainfall that resulted in the loss of basic cations. The acidification of soil may be accelerated by high levels of nitrogen fertilizer application to soil, atmospheric acid deposition and industrial climatic conditions [
2]. When paddy soil pH is <5.0, the availability of phosphorus (P) decreases and rice yield drops sharply. Soil P availability declined due, primarily to factors, such as low pH, high level of active iron (Fe) and aluminum (Al) in acid soils, and phosphate anions reacting with Fe and Al oxides to form insoluble phosphates in certain forms, such as iron-bound phosphate (Fe-P), aluminum-bound phosphate (Al-P) and occluded-phosphate (O-P). Phosphate anion is precipitated by calcium (Ca) to form calcium-bound phosphate (Ca-P) in alkaline soils [
3]. Pi components combined with Fe, Al and Ca are not easily used by plants. However, soil Al-P, Fe-P and some Ca-P can be converted into free phosphate, which provides an important P pool for plant growth [
4]. The solubility of the gel film on the surface of O-P molecules is extremely low, resulting in insufficient utilization of O-P by plants.
Roots exhibit adaptive strategies under low P availability by changing root morphology and physiology. Changes in root morphology can promote root–soil contact, which is particularly significant in P acquisition from soils [
5]. In addition, roots secreted organic acids and P-solubilizing enzymes and increased the expression of high-affinity P transporters in order to cope with P deficiency, as well as Al toxicity [
6]. The root system of rice triggers the secretions of organic anions in response to P deficiency in acidic soils [
7,
8]. However, adequate research on the effects of this process on the acquisition of P is still lacking. The organic acid anions secreted by rice roots are mediated by transporters that are the part of the multidrug and toxic compound extrusion protein family.
OsFRDL4 is capable of transporting citrate and was activated in the presence of Al
3+ [
9]. After mobilization at the rhizosphere, P is absorbed by roots as Pi (H
2PO
4−), which is regulated by membrane-spanning phosphate transporter proteins [
10]. Pi anions across the membrane are driven by plasma membrane H
+-ATPase by consuming ATP. High-affinity Pi transporters (the
Pht1 family) are induced under phosphate deficiency, particularly in the roots [
11].
Biochar is produced by the pyrolysis of biomass residue in anoxic and high-temperature environments, which was found to be a better soil improvement material than other amendments [
12,
13]. Rice-residue biochar has been reported to improve the P availability and P use efficiency of applied or reserve P to meet current P needs [
14]. Biochar increases the available Pi component in the soil and maintains high soil P content for at least 2 years after application [
15]. The positive effect of biochar on soil fertility is related to its high surface area and cation exchange capacity. These properties mean that biochar increases the water-holding capacity of soil and prevent N and P from being leached from soil [
16,
17]. Biochar changes the physicochemical properties of soil (such as the pH, porosity and redox state) and improves nutrient mobilization [
18,
19]. Biochar is alkaline, and it can improve soil fertility by maintaining or increasing soil pH. Biochar can be used as an alternative to lime to amend acidic soil. Meta-analyses have shown that the increase in productivity of biochar-amended acidified soil is mainly caused by the “liming effect” [
20]. Increasing the soil pH can decrease Al bioavailability and the fixation of P by Fe and Al cations (Fe
3+ and Al
3+) [
21,
22]. The surface P adsorption and the adsorption rate of ferrihydrite were decreased in the presence of biochar [
23]; however, the desorption performance was improved when the ferrihydrite was combined with biochar. Therefore, biochar can reduce the adsorption of P by metallic oxides and enhance the availability of P in acidic soils.
Previous studies have shown that biochar has significant indigenous effects on soil properties and crop yields [
24]. However, few studies have analyzed the effect of common acidic soil amendments (biochar and P fertilization) on the adaptive response of rice at the rhizosphere level. The present study focused on (a) the effects of adding biochar on soil P fraction and (b) the subsequent effect of rhizosphere changes induced by the amendments on adaptive responses, such as the changes in citric acid exudation and the expression levels of related efflux transporters. In addition, this study examined the phosphate transporter-mediating effect of rice roots on Pi uptake.
4. Discussion
Biochar has been found to play an important role in improving the availability of P in soil [
34,
35]. Studies have shown that biochar application increases soil pH and decreases the exchangeable acidity and exchangeable Al content [
36,
37,
38]. The results of the present study indicated that applying biochar markedly affected the growth of rice plants (
Table 5). Applying biochar and washed biochar increased the rice plant biomass by 36% and 9.8%, respectively, and applying the equivalent lime increased the biomass by 7.3%. A close relationship was found between promoting rice growth and improving soil characteristics. Adding biochar to soil increased the content of available P in the soil. Biochar provided liming and nutrient adsorption effects due to larger quantities of ash and surface area [
39]. Then, biochar relieved other plausible constraints of this severely acidified paddy soil (low pH and highly exchangeable Al), as shown in
Table 2, and, therefore, increased the overall vigor of the rice plants and the physiological responses of the plants to the lack of P (
Table 4 and
Figure 2). The increase in the pH caused by the liming effect of applying biochar may have promoted proton extrusion from the roots, which is the key process involved in P acquisition and the adaptive release of organic anions [
40].
Rice-straw biochar contains P, which could directly affect the Pi fractions in the soil [
35]. However, incorporation of biochar into soil also alters soil pH, the physical and chemical properties of soil, thus, indirectly affecting the soil P concentration. Rice residual biochar directly increases soil P availability by releasing P from biochar itself, or indirectly increases soil P availability by reducing P adsorption [
14]. Orthophosphate and pyrophosphate are the main forms of P in the biochar, such as manure, which can directly increase soil P content [
39]. In the present study, biochar application resulted in an increase in pH, from 4.29 to 4.60, which contributed to P desorption and increased P solubility in the soils (
Table 2). The application of biochar could change the form of P fixation/adsorption agents, such as Fe, Al and Ca, and had a significant effect on the transformation of P in soil [
41]. Applying phosphate fertilizer mainly increased the content of the Fe-P component in the soil (
Table 4). In acidic soils, most added mineral P is easily combined with Fe [
42]. Adding biochar containing relatively large amounts of Ca
2+ and Mg
2+ to soil can cause the formation of phosphate precipitates and increase P adsorption [
43]. The results of the present study indicated that applying biochar increased the Ca-P fraction, which might improve the availability and plant uptake of P in soil (
Figure 1). The washed biochar contained less Ca
2+ than the biochar and was less alkaline. Applying washed biochar decreased the contribution of Ca-P to the total P content in the soil. Mukherjee et al. showed that Ca-P content increased significantly with biochar application rate [
14]. The result of the present study was consistent with that of Ch’ng et al., who also observed that the soil Ca-P fraction increased in biochar-improved soil [
44]. The increase in the Ca-P fraction may be related to the chemistry and retention of Ca [
39]. Occluded P (the surfaces of Fe phosphate particles covered with an Fe
3+ oxide film) is not available to plants. The results indicated that applying biochar decreased the occluded P content when the P content was low (
Table 4). Chathurika et al. reported that biochar addition did not affect the occluded P content in two different soils [
45], while other studies indicated that biochar significantly reduced the occluded P content [
4]. The decrease in occluded P in the present study was accompanied by the marked responses of the roots to a lack of P, including the increase in the citrate exudation rate and the upregulation of the phosphorous transporter gene
OsPT1 (
Figure 2 and
Figure 3).
Depletion of the P content in rhizosphere soil generally indicates the uptake of P [
46]. A significant decrease in the rhizosphere NaHCO
3-P and Al-P fractions was observed in acidic soil, and no changes were observed for rhizosphere Fe-P and Ca-P fractions in comparison with bulk soil (
Table 2 and
Table 4). Biochar application increased the concentration of Al-P component and had no effect on the content of the Fe-P fraction, in both rhizosphere and bulk soil. Bornø et al. found that three different types of biochar had no effect on the content of NaOH-extractable Pi (Fe-P and Al-P) in bulk soils [
47]. However, the NaOH-Pi fraction in rhizosphere soil increased significantly after biochar treatment without P supply. Mukherjee et al. reported that the content of Al-P and Fe-P increased significantly with an increase in biochar application, regardless of the initial total P state of the test soil [
14], while Hong et al., showed that biochar could significantly increase Al-P content and decrease Fe-P content [
48]. These studies demonstrated that the effects of biochar on P fractions in soil depended on the type and quantity of biochar, as well as the soil type. Sparingly soluble inorganic P is not readily accessible to some species (such as grasses) but is available to some legumes [
49]. However, rice has been found to grow well when inorganic P is supplied as AlPO
4 rather than FePO
4, indicating that Al-P is very available to rice [
50]. Root exudates are very important for the activation of sparingly soluble phosphates in soil [
51].
Applying biochar improved rice root growth in the acidified paddy soil, significantly increasing the total root length and root surface area (
Table 5). Biochar amendment resulted in more extensive root systems and improved P uptake in acidic paddy soil [
52]. It has been reported that the whole rice genome contains 13 members of the
Pht1 family [
10], among which
OsPT1,
OsPT2 and
OsPT6 are primarily expressed in the roots of rice and are related to Pi absorption and transportation [
53,
54].
OsPT1, a member of the
Pht1 family, was enhanced and played an important role in P uptake under P deficiency [
55]. The expression level of the phosphate transporter gene
OsPT1 was upregulated in rice roots by the application of biochar under low-P stress. However, the expressions of
OsPT2 and
OsPT6 were downregulated in the roots under biochar amendment, suggesting that the Pi uptake through these phosphate transporters was downregulated by plant P status (
Figure 3). The release of P was extremely dependent on high concentrations of organic acids. Among them, citrate is very effective in the mobilization of inorganic P, followed by malate, oxalate and tartrate [
56]. The results showed that the root efflux of the citrate anion was enhanced by the application of biochar in the P-deficient rice plants, along with the increased expression of
OsFRDL4 under low-P and acidic soil conditions (
Figure 2 and
Figure 3). The protein encoded by
OsFRDL4 is closely related to citric acid transport, and the amount of organic acid secreted is proportional to the
OsFRDL4 expression [
9]. The over-expression of enzymes responsible for producing organic acids in roots increases Pi uptake from soil with a high Al content or a P deficiency [
57,
58].