New Insight into Phosphorus Release of Rhizosphere Soil in the Water Level Fluctuation Zone

Abstract

Many phosphorus release experiments have been conducted in the water level fluctuation zone (WLFZ). However, the effects of rhizospheres on the risk of soil phosphorus release remain unclear. In this study, twenty-four soil samples were collected from the WLFZ of the Pengxi River, the largest tributary of the northern Three Gorges Reservoir. Physicochemical properties, phosphorus forms and phosphatase activity in rhizosphere and near-rhizosphere soils were investigated. Both the total phosphorus (TP) and water-extracted organic phosphorus (H₂O-P_o) contents were significantly higher in rhizosphere soils than in near-rhizosphere soils (p < 0.05). Enrichment rates (ER) of TP and H₂O-P_o were 42.4% and 180.3%, respectively. Significant increases were also found in activities of acid phosphatase, alkaline phosphatase, and phosphodiesterase in the rhizosphere, and corresponding ERs were 95.3%, 76.1%, and 22.0%, respectively. Environmental factors, such as pH, organic matter, and amorphous manganese, made significant contributions to the variations of phosphorus forms and phosphatase activities. The risk of phosphorus release to water in the rhizosphere soils of the study area increased slightly, according to the water-extracted phosphorus index. This risk may further increase due to flooding, as plant uptake of soil phosphorus would be inhibited while inorganic phosphorus is still generated by phosphatase.

1. Introduction

Most of the surface water in the world is facing the problem of eutrophication, and phosphorus is one of the factors causing this [1]. The water level fluctuation zone (WLFZ) is the land exposed when water levels in rivers, lakes, and other water bodies lower. It is a kind of aquatic–terrestrial ecotone. Periodic exposure and submergence result in a large amount of matter exchange between the WLFZ and the water body [2]. Usually, the WLFZ is an important area for phosphorus deposition and an important source of phosphorus to adjacent water bodies [3].

The Three Gorges Reservoir (TGR) is a hydroelectric reservoir formed after the completion of the Three Gorges Dam. After successful impoundment to store water at the target 175 m above sea level, the total area of the TGR WLFZ reaches 348.9 km² [4]. The water level of the TGR is regulated to be between 145 and 175 m, and hence the height range of the WLFZ is 30 m. An anti-seasonal operational mode is used for water level management in the TGR so that low water levels are maintained from early April to early September for flood control, and high water levels are maintained from mid-September to the end of March of the following year for power generation.

Hence, in summer, a large area of the WLFZ is exposed, with relatively greater areas where the reservoir banks exhibit gentler foreshore slopes. During this period, the weather is warm and wet, and light and nutrients are sufficient for good plant growth. More than 75% of the area of the TGR WLFZ has vegetation cover during the exposure period. Most of this is naturally grown as artificial cultivation of crops was prohibited in the WLFZ. Frequent flooding-drying cycles can lead to a reduction in biodiversity in the WLFZ. However, bermudagrass (Cynodon dactylon (L.) Pers.) experiences a competitive advantage after several consecutive high-intensity flooding episodes [5].

The rhizosphere is a key region for interactions between plants, soil, and microbes [6]. Plant roots can significantly change the microenvironment in the rhizosphere through various physiological activities [7], and these can affect the transformation of soil phosphorus. Studies have shown that plant-induced rhizosphere effects can affect soil phosphorus form transformation and phosphatase activity in wetlands [8], grasslands [9] and farmlands [10]. Additionally, environmental factors in the WLFZ such as elevation, flooding time, and UV irradiation can also influence soil phosphorus form transformation [3,11,12]. However, to date there has been little discussion of the effects of plant growth on soil phosphorus form transformation in the WLFZ.

It therefore remains unclear whether the rhizosphere in the WLFZ will enrich phosphorus and affect phosphorus forms, and possibly change the risk of the release of soil phosphorus into the WLFZ. Plant growth environments in the WLFZ are quite different from those in grasslands and farmlands owing to the periodic water level fluctuations. The transformation of phosphorus forms in rhizosphere soils in the WLFZ may change the possibility of soil phosphorus release, and hence affect water quality during flooded periods.

Soil phosphatases are a class of extracellular enzymes that can catalyze the hydrolysis of the phosphate group and widely exist in soils [13]. They have been found to regulate the mineralization of organic phosphorus and phosphorus turnover, and significantly increase the content of available phosphorus in soil [14,15]. Soil phosphatase activities are related to soil phosphorus forms [16], and can be affected by plant roots and bacterial communities [9,10]. So far, studies of soil phosphorus transformation in the WLFZ have focused mainly on the change in phosphorus concentrations under different classifications of phosphorus forms, or on phosphorus release to the water [4,11]. Hence, phosphatase activities and the phosphatase-mediated phosphorus transformation process have so far been largely ignored. Therefore, in this study the phosphorus transformation process in rhizosphere soils in the WLFZ is analyzed and compared to processes in near-rhizosphere soils.

Previous studies of soil phosphorus form in the WLFZ have mainly focused on the determination of inorganic phosphorus, organic phosphorus, and total phosphorus using standard SMT extraction methods [17,18] and on the soil available phosphorus using the Olsen extraction method [19,20]. However, in this study different forms of both organic and inorganic phosphorus are determined using separate tests after each extraction step in the Hedley continuous extraction method [21], providing an in-depth analysis of soil phosphorus form distribution and transformation in the WLFZ.

This study aims to: (1) investigate the various phosphorus form distributions and influencing factors in rhizosphere/near-rhizosphere soils in the WLFZ, TGR; (2) investigate the effects of the rhizosphere on phosphatase activity and phosphatase-mediated soil phosphorus transformation in the WLFZ; (3) assess the risk of phosphorus release in WLFZ rhizosphere/near-rhizosphere soils.

2. Materials and Methods

2.1. Study Area

The Pengxi River (31°00′–31°42′N, 107°56′–108°54′E) is a first-class tributary of the Yangtze River, with the length of 182.4 km and drainage area of 5172.5 km². The tributary is 247 km away from the Three Gorges Dam, approximately in the center of the TGR. The Pengxi River Basin is a low mountainous and hilly area in the eastern part of the Sichuan Basin, with purple soil and the yellow loam soil as the main soil types [22]. Soil particles are mainly silt, with an additional small and varying sand and clay contribution. The climate is subtropical humid monsoon, with most rainfall occurring during the summer. Annual mean air temperature and precipitation are 18.2 °C and 1053 mm, respectively [23].

Among all the tributaries to the TGR, the Pengxi River Basin has the largest WLFZ, with an area of approximately 56.6 km², occupying 16.3% of the total WLFZ TGR. Plants are naturally grown without fertilizer addition and dominated by annual herbs. Vegetation cover and flooding times differ throughout the Pengxi River WLFZ and vary with height above the lowest water level datum (i.e., 145 m altitude). The general pattern is the lower the height, vegetation cover is less and flooding times are longer. Normally, the WLFZ between 10 m and 20 m above the low water datum (i.e., 155 m to 165 m altitude) experiences frequent water level fluctuations during the plant growth period. The area above a height of 30 m above the low water datum (i.e., 175 m altitude) is outside the WLFZ and experiences no flooding.

2.2. Sample Collection and Processing

Six sampling sites were arranged within approximately 30 km from the end of the backwater to the tributary (Figure 1), namely the Qukou (QK), Yanglu (YL), Quma (QM), Gaoyang (GY), Huangshi (HS) and Shuangjiang (SJ) sampling sites. All sites were on foreshores which gently sloped and were covered mainly by bermudagrass (Cynodon dactylon (L.) Pers.). The soil particle size was determined by laser particle size analyzer (Mastersizer 3000; Malvern Panalytical, UK). The soil samples were composed with 30.89–35.43% of sand, 51.81–59.52% of silt and 6.23–11.07% of clay. There was no significant difference in soil particle size composition at each site. The geographical environments of the different sampling sites along the reservoir tributary exhibited little difference (Table S1). Samples of rhizosphere and near-rhizosphere soils within the WLFZ were collected between elevations of 155 m and 165 m in 2019, with a total of 24 samples. Each sample consisted of a mixture of 3–5 sub-samples collected in the vicinity of each sampling site in order to account for possible small-scale variability in soil properties.

During sampling, only soil with intact root systems was excavated. In order to eliminate the effect of spatial heterogeneity, referring to the sampling methods provided by previous studies [24,25], near-rhizosphere soil was dislodged by shaking the roots gently, while the soil remaining adhered to the root was regarded as rhizosphere soil.

2.3. Physiochemical Properties

The pH value of a soil sample was determined using an electrode-based method with a 1:2.5 water–soil ratio. The content of soil organic matter (OM) was determined by mass loss after calcination at 550 °C for 5 h following the loss-on-ignition method [26]. The contents of amorphous iron oxide (Fe_ox) and amorphous manganese oxide (Mn_ox), extracted using an ammonium oxalate buffer solution, were determined using an iCAP-6300 Duo inductively coupled plasma optical emission spectrometer (Thermo, Waltham, MA, USA).

The fumigation-extraction method was used to determine the content of soil microbial biomass phosphorus (P_mic) [27]. Details of the extraction are given in the Supplementary Materials (Text S1). Soil microbial phosphorus concentration (C_pmic) was estimated using the following formula:

C_pmic = (C_pa − C_pb)/0.4,(1)

where C_pa is the phosphorus concentration extracted after fumigation (mg/kg), and C_pb is the phosphorus concentration extracted without fumigation (mg/kg).

2.4. Phosphorus Content and Forms

The contents of inorganic phosphorus (IP), organic phosphorus (OP) and total phosphorus (TP) in the WLFZ soil samples were separated using the SMT extraction method developed under the framework of the European Standards Testing Committee [28], and analyzed using the phosphorus molybdenum blue colorimetric method [29]. Specific steps are shown in Figure S1.

A total of 7 phosphorus forms were determined using the modified Hedley grading extraction method [30]. Inorganic phosphorus forms were divided into unstable water-extracted inorganic phosphorus (H₂O-P_i), moderately unstable sodium bicarbonate extracted inorganic phosphorus (NaHCO₃-P_i), moderately resistant sodium hydroxide extracted inorganic phosphorus (NaOH-P_i), and highly resistant hydrochloric acid extracted inorganic phosphorus (HCl-P_i). Organic phosphorus forms were divided into unstable water-extracted organic phosphorus (H₂O-P_o), moderately unstable sodium bicarbonate extracted organic phosphorus (NaHCO₃-P_o), and highly resistant sodium hydroxide extracted organic phosphorus (NaOH-P_o).

The steps of the phosphorus grading and sequential extraction method are summarized in Figure 2. A 0.45 μm water-based syringe filter was used on the extraction after each step. The total phosphorus was then determined using the phosphorous molybdenum blue colorimetric method after 5% potassium persulfate digestion [31]. Inorganic phosphorus in the filtrates was directly measured using the phosphorous molybdenum blue colorimetric method [32]. The content of organic phosphorus was calculated as shown in Figure 2.

2.5. Phosphatase Activity

The activities of soil phosphatases including acid phosphatase (ACP), alkaline phosphatase (ALP), phosphodiesterase (PDE) and phytase (PAE) were determined. ACP and ALP are phosphomonoesters, catalyzing the hydrolysis of low-molecular-weight phosphorus compounds with monoester bonds [31]. PDE can catalyze the hydrolysis of low-molecular-weight phosphorus compounds with diester bonds [33]. PAE can catalyze the hydrolysis of phytic acid and its salts, and generate inositol and phosphoric acid (or phosphate) [32].

The activities of ACP, ALP and PDE were characterized by the amount of p-nitrophenol (pNP) produced by the enzymatic reaction of ACP, ALP and PDE per gram soil mass per hour [34]. The activity of PAE was determined using the ammonium metavanadate molybdenum yellow method [35], characterized by the amount of inorganic phosphorus produced by the enzymatic reaction of PAE per gram soil mass per hour. Details of phosphatase activity determination are described in the Supplementary Materials (Text S2), and the measurement conditions are summarized in Table S2.

2.6. Enrichment Rates

The rhizosphere-enrichment rate (ER) was used to characterize the enrichment effect of the rhizosphere on substances [35], as follows:

ER_i = (C_R,i − C_NR,i)/C_NR,i × 100%,(2)

where C is concentration; i represents Fe_ox, Mn_ox, each phosphorus form, or phosphatase activity; and R and NR represent rhizosphere and near-rhizosphere soils, respectively.

2.7. Data Analysis

One-way ANOVA was used to analyze the differences of phosphorus content and phosphatase activity between rhizosphere and near-rhizosphere soils. Pearson’s correlation analysis was used to analyze the correlation between soil physical and chemical properties, P forms, and phosphatase activities. Redundancy analysis (RDA) was used to determine the main environmental factors affecting the distribution of P forms in rhizosphere and near-rhizosphere soils. One-way ANOVA and Pearson’s correlation analyses were performed using the SPSS 20 software. RDA was performed using canoco5 software. Graphs were created using Origin 2021 software.

3. Results

3.1. Physicochemical Properties of WLFZ Rhizosphere/Near-Rhizosphere Soils

The physical and chemical properties of WLFZ rhizosphere/near-rhizosphere soils are summarized in Figure 3. The mean pH values of rhizosphere and near-rhizosphere soils in the WLFZ were 6.95 ± 0.23 and 6.98 ± 0.24, respectively, showing no significant difference (p > 0.05). However, pH values in rhizosphere soils were decreased compared to near-rhizosphere soils at all sampling sites except at the GY site (Table 1). The mean concentrations of Fe_ox and Mn_ox in the rhizosphere soils were 3.32 ± 0.67 and 0.78 ± 0.22 g/kg, respectively, while those in the near-rhizosphere soils were 2.73 ± 0.77 and 0.31 ± 0.09 g/kg, respectively. Hence, both Fe_ox and Mn_ox had significantly higher contents in the rhizosphere soils (p < 0.05).

The mean concentrations of OM and P_mic in the rhizosphere soils were 83.00 ± 19.34 g/kg and 21.47 ± 5.11 mg/kg, respectively, while those in the near-rhizosphere soil samples were 70.73 ± 13.45 g/kg and 15.64 ± 7.86 mg/kg, respectively. Hence, concentrations of OM and P_mic were also significantly higher in the rhizosphere soils (p < 0.05).

Since the change in soil phosphorus form and phosphorus release are related to the location [4], the spatial differences between WLFZ rhizosphere/near-rhizosphere soil contents were considered. However, in this study the physicochemical properties do not show significant differences between sites (Table 1). In general, no spatial differences were found in the WLFZ rhizosphere/near-rhizosphere soils along the Pengxi River tributary of the TGR.

3.2. Influence of Rhizosphere on Phosphorus Contents and Forms in the WLFZ

The phosphorus contents of WLFZ rhizosphere/near-rhizosphere soils extracted by the SMT method are shown in Figure 4a. The concentrations of TP, IP, and OP in rhizosphere soils in the WLFZ were 854.6 ± 111.61, 441.8 ± 46.83, and 166.0 ± 51.44 mg/kg, respectively. Those in near-rhizosphere soils were 600.3 ± 94.23, 483.8 ± 50.40, and 169.0 ± 30.35 mg/kg, respectively. Different phosphorus forms showed different rhizosphere-enrichment capacities. The ER_TP was 42.37%, showing an obvious rhizosphere enrichment (p < 0.05). The IP showed an exactly reverse effect, with significantly lower concentrations in the rhizosphere (p < 0.05).

No significant spatial difference was found in TP, IP, and OP concentrations of different sites along the Pengxi River in the WLFZ rhizosphere/near-rhizosphere soils (Table 2).

The phosphorus forms of WLFZ rhizosphere/near-rhizosphere soils extracted by the Hedley method are shown in Figure 4b,c. The concentrations of H₂O-P_i, NaHCO₃-P_i, NaOH-P_i, and HCl-P_i in rhizosphere soils in WLFZ were 2.44 ± 0.99, 34.73 ± 14.21, 53.19 ± 19.96, and 317.6 ± 34.05 mg/kg, respectively. Additionally, corresponding concentrations in near-rhizosphere soils were 3.60 ± 1.20, 43.46 ± 11.74, 53.23 ± 25.07, and 352.1 ± 51.97 mg/kg, respectively. All inorganic phosphorus forms showed lower concentrations in the rhizosphere in WLFZ, but only the differences in H₂O-P_i and HCl-P_i were significant (p < 0.05) (Figure 4b).

The concentrations of H₂O-P_o, NaHCO₃-P_o, and NaOH-P_o in rhizosphere soils in WLFZ were 3.28 ± 1.30, 4.57 ± 2.31, and 24.83 ± 11.09 mg/kg, respectively. Additionally, those in near-rhizosphere soils were 1.17 ± 0.75, 4.91 ± 3.91, and 28 ± 9.65 mg/kg, respectively. The ER of H₂O-P_o was 180.3%, showing a significant enrichment in the rhizosphere (p < 0.05). No significant differences were found in the contents of NaHCO₃-P_o and NaOH-P_o between rhizosphere and near-rhizosphere soils, though the former was slightly lower (Figure 4c). The compositions of different phosphorus forms at each site are shown in Figure 4d,e.

Generally, no significant spatial difference in phosphorus forms extracted by the Hedley method in the WLFZ rhizosphere/near-rhizosphere soil was found among different sites along the Pengxi River.

3.3. Influence of Rhizosphere on Phosphatase Activity in WLFZ

The phosphatase activities of the WLFZ rhizosphere/near-rhizosphere soils are summarized in Figure 5. The activities of ACP, ALP, PDE, and PAE in the WLFZ rhizosphere soils were 3.75 ± 1.95 μmol pNP/(g h), 6.18 ± 2.83 μmol pNP/(g h), 0.72 ± 0.18 μmol pNP/(g h), and 11.36 ± 0.68 μmol P/(g h), respectively. Those in near-rhizosphere soils were 1.91 ± 1.02 μmol pNP/(g h), 3.51 ± 1.29 μmol pNP/(g h), 0.59 ± 0.16 μmol pNP/(g h), and 11.18 ± 0.99 μmol P/(g h), respectively. Hence, ACP, ALP and PDE showed significantly higher activities in the rhizosphere soils (p < 0.05). The ER of ACP, ALP, and PDE were 95.34%, 76.07%, and 22.03%, respectively, showing obvious rhizosphere-enrichment effects.

Similar to the phosphorus forms, spatial differences in the phosphatase activities in WLFZ rhizosphere and near-rhizosphere soils were not significant between the different sites along the Pengxi River TGR tributary (Figure 5b,c).

3.4. Relationship between P Forms/Phosphatase Activity and Environmental Factors

Redundancy analysis showed that pH (n = 24, F = 3.0, p < 0.01), Mn_ox (n = 24, F = 2.1, p < 0.01), and P_mic (n = 24, F = 1.6, p < 0.05) made significant contributions to the variation of phosphorus forms in the WLFZ rhizosphere/near-rhizosphere soils (Figure 6a). OM (n = 24, F = 29.4, p < 0.01), pH (n = 24, F = 8.9, p < 0.01), and Mn_ox (n = 24, F = 7.0, p < 0.05) made significant contributions to the variation of phosphatase activities in the WLFZ rhizosphere/near-rhizosphere soils (Figure 6b).

The correlations between physicochemical characteristics, phosphorus forms and phosphatase activities in the WLFZ rhizosphere/near-rhizosphere soils are shown in Figure 6c. The H₂O-P_o content was significantly and negatively correlated with pH (p < 0.01). The contents of TP and OP in the WLFZ soils were significantly and positively correlated to the activities of ACP, ALP and PDE (p < 0.01). The activities of ACP, ALP and PDE in WLFZ soils were all significantly and positively correlated to OM concentration (p < 0.01).

Further correlation analyses showed that, in the near-rhizosphere soils (Figure S2b), PDE activity was significantly (p < 0.01) and negatively correlated with NaHCO₃-P_i and NaOH-P_i, while ALP activity was significantly (p < 0.01) and negatively correlated with NaOH-P_i. No significant correlation was found in rhizosphere soils (Figure S2a).

4. Discussion

4.1. Phosphorus Form Transformation in Rhizosphere Soil

In the rhizosphere, the exchange of substances between plants, microorganisms and soil is active and frequent. As a nutrient element for plant and microbial growth, soil phosphorus tends to migrate to the rhizosphere due to the existence of plant roots and the accumulation of root microorganisms [15]. Rhizosphere-enrichment effects lead to the increase in TP content.

The oxidation reduction potential (ORP) plays an important role in phosphate speciation and availability. The oxygen transport tissues from leaves to roots and oxygen released from roots to surrounding areas can significantly increase the ORP of rhizosphere [36,37]. A higher ORP is beneficial to phosphate immobilization and reduce phosphorus availability by promoting the production of precipitation and adsorption [38]. Additionally, a concentration gradient of dissolved phosphorus can be formed near the rhizosphere, which induced the movement to the roots.

At the same time, phosphorus in soil, especially the inorganic phosphorus component is readily directly absorbed and utilized by plant roots as nutrient [7], which may be the reason for the significant observed decrease in the IP content of the rhizosphere soil. Furthermore, H₂O-P_i and HCl-P_i are more easily absorbed and utilized by plants, and this may have caused the decreased concentrations in the rhizosphere soils.

The reduction in inorganic phosphorus in rhizosphere soils can also be affected by microbial activity. Due to the rhizosphere-enrichment effect, the amount of soil microorganisms in rhizosphere was more abundant and the microbial activities were higher [39]. This may lead to an increased consumption of P for microbial growth. Additionally, the phosphatase secreted by microorganisms will increase with the abundance enrichment of microorganisms [40], which resulted in more active phosphorus. In addition, microorganisms may stimulate P utilization by plants through multiple mechanisms, such as the production of plant hormones and quorum sensing [41].

The enrichment of H₂O-P_o was particularly obvious in the rhizosphere samples. On the one hand, H₂O-P_o is a weakly adsorbed organic phosphorus [42], which is the most likely component of OP to accumulate in the rhizosphere of plants. On the other hand, monoester phosphorus is the main component of H₂O-P_o [11,43,44], and the rhizosphere has an obvious aggregation effect on phosphomonoesterases (including ALP and ACP). Such enzymes play an important role in the conversion of H₂O-P_o.

The sum of H₂O-P_i, NaHCO₃-P_i and NaOH-P_i can be expressed as bioavailable inorganic phosphorus (Bio-P_i). In general, they are considered as the bioavailable part of soil inorganic phosphorus, which were also easily released [4]. The sum of H₂O-P_o, NaHCO₃-P_o and NaOH-P_o can be expressed as bioavailable organic phosphorus (Bio-P_o). They are organic phosphates in the soil that can be enzymatically hydrolyzed [11]. The concentrations of Bio-P_i and Bio-P_o in the rhizosphere soils in the WLFZ were 90.36 ± 34.20 and 34.65 ± 12.18 mg/kg, respectively. Those in near-rhizosphere soils were 100.29 ± 34.68 and 34.35 ± 12.01 mg/kg, respectively. Hence, the inorganic phosphorus in the WLFZ rhizosphere soil was decreased slightly due to the regulation of phosphatase activities and the enhancement of inorganic phosphorus utilization.

pH was the main factor affecting soil phosphorus form transformation in the WLFZ. pH will reduce in rhizosphere because plant roots secreted organic acids such as citric acid and oxalic acid [45]. Rhizosphere acidification was observed in this study. Rhizosphere acidification can influence element mobilization at the micro-scale through adsorption or precipitation [46]. The solubility of P can increase with pH decreasing and acidification enhanced the activation of bound P [47]. Additionally, organic anion concentrations can also make contributions to soil P availability and plant uptake [48].

The enrichment effects on phosphorus by plant roots have also been observed in grasslands [49] and mountainous ecosystems [50]. However, different plants show different preferences to rhizosphere-enrichment effects. For example, red sandalwood (Pterocarpus indicus Willd.) in the mountainous area of Yunnan, China, showed a preference for the medium active phosphorus form NaOH-P [50]. In contrast, Ruzigrass (Urochloa ruziziensis (R. Germ. and C.M. Evrard.) Morrone and Zuloaga) grown in Botucatu, Brazil, showed significant enrichment of NaHCO₃-P and NaOH-P [51]. The bermudagrass observed in this study in the WLFZ of the Pengxi River was associated with an obvious enrichment of H₂O-P_o.

4.2. Phosphatase Enrichment in Rhizosphere Soil

There are two main sources of phosphatase in soil. One is the active secretion from plants, bacteria, and fungi. The other is the release from decaying animal and plant residues [52,53,54,55]. Soil phosphatases secreted by microorganisms and plants are crucial to the transformation process of various phosphorus forms [56]. Phosphatases are related to the bioavailability of soil phosphorus, as this extracellular enzyme can decompose organic phosphorus into inorganic phosphorus directly utilized by animals and plants.

ACP, ALP and PDE showed obvious rhizosphere-enrichment effects. The phosphatase secreted by both plant roots and high-density microbial populations in the rhizosphere soil region was an important reason for the rhizosphere-enrichment effect. In addition, the adhesive film of plant roots can effectively retain the phosphatase secreted by plant roots [7], and root hairs can promote phosphatase activities in the rhizosphere [57].

OM and Mn_ox were the main factors affecting soil phosphatase activities in the WLFZ. Labile organics are continuously secreted into the soil by plant roots during growth, and during the decay of old plant roots and surface exfoliation gathered in the rhizosphere [58,59]. Root exudates and plant residues led to a significant increase in the OM concentration in the rhizosphere soil of the WLFZ. OM in the soil can provide abundant carbon sources and minerals, and promote the growth of plants and microorganisms, hence the activities of secreted phosphatase are improved [21]. In a wetland with environment similar to this study, OM was also found to play an important role in the rhizosphere enrichment of phosphomonoesterases and phosphodiesterases [15]. In addition, plant roots can form colloidal films of iron or manganese oxides [60], and phosphatases can be stably adsorbed on manganese oxide particles with good surface activity in soil [61]. Some mineral surfaces were confirmed to provide adsorption sites for phosphatase [62].

The activity of extracellular phosphatase in soil has been reported to be negatively correlated with the availability of soil inorganic phosphorus [63]. In this study, inorganic phosphorus supply in near-rhizosphere soils may have been enough to trigger negative feedback and inhibit some phosphatase secretion, thus resulting in the observed significant negative correlations between inorganic phosphorus and phosphatase activities. However, in the rhizosphere soils, inorganic phosphorus may have been consumed due to the absorption and utilization by plant roots. Phosphatase activities were maintained, as plants and microorganisms can excrete various phosphatases in response to environmental change when the content of bioavailable phosphorus decreases in the rhizosphere [20].

4.3. Implication

Water-extracted phosphorus (H₂O-P) is a convenient indicator for predicting the risk of soil phosphorus release suitable for various soil types and land use types [64,65,66]. The mean contents of H₂O-P in the WLFZ rhizosphere soils and near-rhizosphere soils found in this study were 5.72 and 4.77 mg/kg, respectively. The general alert H₂O-P concentration in the WLFZ soils was 8.5 mg/kg [64,67,68]. Hence, the overall soil phosphorus release risk in the WLFZ was relatively low.

Wang et al. [4] pointed out that the phosphorus release of WLFZ in the estuary of tributaries in the TGR should be monitored. Zhou et al. [3] found that soil erosion was the main reason for phosphorus loss in this area. Our results suggested that the rhizosphere-enrichment effect might slightly increase the risk of soil P release in the WLFZ, especially H₂O-P_o, the weakly adsorbed organic form. The increase in phosphatase activity in the rhizosphere soil can accelerate the mineralization of organic phosphorus. The absorption and utilization by plants promoted inorganic phosphorus consumption. Compared with the consumption of H₂O-P_i, the more significant enrichment of H₂O-P_o in the rhizosphere eventually led to the increase in the H₂O-P level. However, the rhizosphere soil may be responsible for higher P release when plants stop inorganic phosphorus consumption during flooding.

The risk of phosphorus release from WLFZ soils was closely related to the prevention and control of water eutrophication [1]. Water level fluctuations remained constant in the WLFZ, even during the plant growth period. In the TGR, periods of flooding can sometimes last so long that they can cause plant death [12]. During such flooding, active phosphorus is more easily released into the water body, as plant growth and phosphorus utilization are inhibited and microorganisms continue secreting phosphatase. Phosphatase activities in the WLFZ have been shown to decrease with the increase in flooding time [69]. The risk of phosphorus release from the WLFZ rhizosphere soils may further increase during flooding. Therefore, the risk of phosphorus release from the rhizosphere soil in the WLFZ should not be ignored during periods of water level fluctuation, especially in the initial stage of prolonged flooding.

In summary, the rhizosphere promoted soil phosphorus mobilization, and slightly increased the risk of phosphorus release from the WLFZ soil of the TGR. Considering the requirement for the prevention and control of water eutrophication, plants could be harvested before long-term flooding (i.e., high water levels) occurs. In order to maintain and utilize phosphorus in the soil, commonly occurring plants that occupy the WLFZ of the TGR should be retained during dry periods (i.e., low water levels).

5. Conclusions

The effects of the rhizosphere on the contents of various phosphorus forms and phosphatase activities were analyzed. The risk of phosphorus release in the rhizosphere soil of the WLFZ was evaluated. The plant growth in WLFZ resulted in significant enrichment of TP, H₂O-P_o, ACP, ALP and PDE in the rhizosphere soils of the TGR WLFZ. Rhizosphere effects changed specific environmental factors such as pH, OM, and Mn_ox, and further affected soil P forms and phosphatase activities. H₂O-P_o presented the highest ER in the rhizosphere soils (180.3%), which was due to its high activity and the enrichment of phosphomonoesterases. The rhizosphere slightly increased the potential risk of soil phosphorus release, as indicated by an increase in the H₂O-P index caused by H₂O-P_o. Therefore, the effect of the rhizosphere on soil phosphorus transformation should not be ignored in the TGR WLFZ. From the perspective of avoiding excessive phosphorus release in the WLFZ, local plants with a high rhizosphere-enrichment rate could be retained to maintain and utilize soil phosphorus effectively during the low water level stage, and properly harvested before long-term flooding.

Author Contributions

Conceptualization, F.F. and J.G.; methodology, Z.T.; software, C.W.; validation, Y.J., F.F. and J.G.; formal analysis, Z.T.; investigation, R.Z.; data curation, C.W.; writing—original draft preparation, Z.T. and Y.J.; writing—review and editing, F.F.; supervision, J.G. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures and Tables

Figure 1. Sampling sites in the water level fluctuation zone of the Pengxi River in the Three Gorges Reservoir.

Figure 2. Procedures of modified Hedley phosphorus grading and sequential extraction method.

Figure 3. The physicochemical characteristics of rhizosphere and near-rhizosphere soils in the water level fluctuation zone: (a) pH, amorphous iron oxide (Feox) and amorphous manganese oxide (Mnox); (b) microbial biomass phosphorus (Pmic) and organic matter (OM). Different letters on the bars indicate the significance level at p < 0.05 between rhizosphere and near-rhizosphere soils.

Figure 4. Phosphorus concentrations extracted by the SMT and Hedley methods: (a) total phosphorus (TP), inorganic phosphorus (IP) and organic phosphorus (OP) extracted by the SMT method; (b) inorganic phosphorus forms extracted by the Hedley method; (c) organic phosphorus forms extracted by the Hedley method, (d) site-specific inorganic phosphorus forms extracted by the Hedley method; (e) site-specific organic phosphorus forms extracted by the Hedley method. R represents the rhizosphere and NR represents the near-rhizosphere. Different letters on the bars indicate the significance level at p < 0.05 between rhizosphere and near-rhizosphere soils.

Figure 4. Phosphorus concentrations extracted by the SMT and Hedley methods: (a) total phosphorus (TP), inorganic phosphorus (IP) and organic phosphorus (OP) extracted by the SMT method; (b) inorganic phosphorus forms extracted by the Hedley method; (c) organic phosphorus forms extracted by the Hedley method, (d) site-specific inorganic phosphorus forms extracted by the Hedley method; (e) site-specific organic phosphorus forms extracted by the Hedley method. R represents the rhizosphere and NR represents the near-rhizosphere. Different letters on the bars indicate the significance level at p < 0.05 between rhizosphere and near-rhizosphere soils.

Figure 5. The activities of acid phosphatase (ACP), alkaline phosphatase (ALP), phosphodiesterase (PDE) and phytase (PAE) in rhizosphere and near-rhizosphere soils in the water level fluctuation zone (a) and at different sites (b,c). R represents the rhizosphere and NR represents the near-rhizosphere. Different letters on the bars indicate the significance level at p < 0.05 in rhizosphere and near-rhizosphere.

Figure 6. Redundancy analysis of the effects of environmental factors on phosphorus forms (a) and phosphatase activities (b), and correlation between physicochemical characteristics, phosphorus forms and phosphatase activities (c). Significance levels: ** p < 0.01; * p < 0.05 (n = 24).

Table 1

The physicochemical characteristics of rhizosphere (R) and near-rhizosphere (NR) WLFZ soils at different sites (mean ± standard deviation).

Site	pH		Pmic (mg/kg)		OM (mg/g)		Feox (g/kg)		Mnox (g/kg)
	R	NR	R	NR	R	NR	R	NR	R	NR
QK	6.95 ± 0.15	6.98 ± 0.12	15.40 ± 1.48	7.20 ± 1.19	71.27 ± 4.06	65.15 ± 3.49	3.98 ± 1.01	2.51 ± 0.45	0.85 ± 0.29	0.34 ± 0.11
YL	7.04 ± 0.02	7.09 ± 0.06	23.53 ± 2.86	17.86 ± 0.47	67.60 ± 7.27	60.69 ± 8.83	3.16 ± 0.39	2.32 ± 0.14	0.80 ± 0.10	0.32 ± 0.04
QM	6.65 ± 0.02	6.70 ± 0.01	17.56 ± 3.28	9.75 ± 6.27	72.33 ± 25.19	68.80 ± 14.00	2.77 ± 0.50	1.84 ± 0.97	0.67 ± 0.18	0.27 ± 0.07
GY	6.72 ± 0.06	6.72 ± 0.09	19.58 ± 0.89	15.03 ± 4.75	96.65 ± 1.09	68.20 ± 0.38	3.29 ± 0.25	2.86 ± 0.16	0.61 ± 0.25	0.24 ± 0.10
HS	7.27 ± 0.03	7.31± 0.02	25.75 ± 0.58	21.56 ± 7.64	94.22 ± 12.85	89.00 ± 14.92	3.25 ± 0.21	3.34 ± 0.12	0.91 ± 0.13	0.36 ± 0.05
SJ	7.08 ± 0.04	7.10 ± 0.06	27.01 ± 3.52	22.43 ± 5.05	95.94 ± 14.78	72.54 ± 2.71	3.44 ± 0.39	3.52 ± 0.19	0.85 ± 0.06	0.34 ± 0.02

Table 2

The content of phosphorus in rhizosphere (R) and near-rhizosphere (NR) WLFZ soils at different sites by the SMT method (mg/kg, mean ± standard deviation).

Site	Total Phosphorus		Inorganic Phosphorus		Organic Phosphorus
	R	NR	R	NR	R	NR
QK	880.9 ± 25.17	619.1 ± 13.36	437.9 ± 10.07	485.7 ± 27.68	153.5 ± 2.52	133.4 ± 9.23
YL	795.3 ± 10.00	530.2 ± 26.85	443.0 ± 15.10	435.4 ± 10.91	151.0 ± 11.74	138.4 ± 20.97
QM	929.5 ± 171.14	512.6 ± 121.64	462.9 ± 54.53	491.6 ± 30.20	152.7 ± 85.57	152.7 ± 30.20
GY	864.1 ± 82.21	562.1 ± 13.42	405.2 ± 19.30	435.4 ± 15.94	194.6 ± 33.56	168.6 ± 10.91
HS	748.3 ± 87.25	672.8 ± 48.66	412.8 ± 55.37	490.8 ± 2.52	175.3 ± 46.14	202.2 ± 12.58
SJ	909.4 ± 33.36	704.7 ± 13.42	488.3 ± 21.81	563.8 ± 26.85	188.8 ± 29.36	198.8 ± 10.84

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/su15086635/s1. Figure S1. Procedures of the SMT method. Figure S2. Correlation between physicochemical characteristics, P forms and phosphatase forms in rhizosphere (a) and near-rhizosphere (b) soils. Significance levels: ** p < 0.01; * p < 0.05. Table S1. The coordinate and geography of sampling sites. Table S2. Conditions for determination of phosphatase activities. Text S1. Method of soil microbial biomass phosphorus (P_mic) determination. Text S2. Method of Phosphatase activity determination. References [15,35,70,71,72,73] are cited in Supplementary Materials file.

References

1. Yamaji, N.; Takemoto, Y.; Miyaji, T.; Mitani-Ueno, N.; Oshida, K.; Ma, J.F. Reducing phosphorus accumulation in rice grains with an impaired transporter in the node. Nature; 2017; 541, 92. [DOI: https://dx.doi.org/10.1038/nature20610] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28002408]

2. Wu, Y.; Wang, X.; Zhou, J.; Bing, H.; Sun, H.; Wang, J. The fate of phosphorus in sediments after the full operation of the Three Gorges Reservoir, China. Environ. Pollut.; 2016; 214, pp. 282-289. [DOI: https://dx.doi.org/10.1016/j.envpol.2016.04.029]

3. Zhou, J.; Wu, Y.H.; Wang, X.X.; Bing, H.J.; Chen, Y.; Sun, H.Y.; Zhong, Z.L. Divergent patterns of soil phosphorus discharge from water-level fluctuation zone after full impoundment of Three Gorges Reservoir, China. Environ. Sci. Pollut. R; 2019; 26, pp. 2559-2568. [DOI: https://dx.doi.org/10.1007/s11356-018-3805-1]

4. Wang, C.; Fang, F.; Yuan, Z.Y.; Zhang, R.; Zhang, W.; Guo, J.S. Spatial variations of soil phosphorus forms and the risks of phosphorus release in the water-level fluctuation zone in a tributary of the Three Gorges Reservoir. Sci. Total Environ.; 2020; 699, 134124. [DOI: https://dx.doi.org/10.1016/j.scitotenv.2019.134124]

5. Zhang, Z.Y.; Wan, C.Y.; Zheng, Z.W.; Hu, L.; Feng, K.; Chang, J.B.; Xie, P. Plant community characteristics and their responses to environmental factors in the water level fluctuation zone of the three gorges reservoir in China. Environ. Sci. Pollut. R; 2013; 20, pp. 7080-7091. [DOI: https://dx.doi.org/10.1007/s11356-013-1702-1] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23589274]

6. Li, J.Y.; Yuan, X.L.; Ge, L.; Li, Q.; Li, Z.G.; Wang, L.; Liu, Y. Rhizosphere effects promote soil aggregate stability and associated organic carbon sequestration in rocky areas of desertification. Agric. Ecosyst. Environ.; 2020; 304, 107126. [DOI: https://dx.doi.org/10.1016/j.agee.2020.107126]

7. Hinsinger, P.; Bengough, A.G.; Vetterlein, D.; Young, I.M. Rhizosphere: Biophysics, biogeochemistry and ecological relevance. Plant Soil; 2009; 321, pp. 117-152. [DOI: https://dx.doi.org/10.1007/s11104-008-9885-9]

8. Kandziora-Ciupa, M.; Nadgorska-Socha, A.; Barczyk, G. The influence of heavy metals on biological soil quality assessments in the Vaccinium myrtillus L. rhizosphere under different field conditions. Ecotoxicology; 2021; 30, pp. 292-310. [DOI: https://dx.doi.org/10.1007/s10646-021-02345-1] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33496898]

9. Cabugao, K.G.; Yaffar, D.; Stenson, N.; Childs, J.; Phillips, J.; Mayes, M.A.; Yang, X.J.; Weston, D.J.; Norby, R.J. Bringing function to structure: Root-soil interactions shaping phosphatase activity throughout a soil profile in Puerto Rico. Ecol. Evol.; 2021; 11, pp. 1150-1164. [DOI: https://dx.doi.org/10.1002/ece3.7036] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33598120]

10. Zhang, L.; Peng, Y.; Zhou, J.C.; George, T.S.; Feng, G. Addition of fructose to the maize hyphosphere increases phosphatase activity by changing bacterial community structure. Soil Biol. Biochem.; 2020; 142, 107724. [DOI: https://dx.doi.org/10.1016/j.soilbio.2020.107724]

11. Gao, J.M.; Chen, Z.M.; Wang, C.; Fang, F.; Huang, J.J.; Guo, J.S. Bioavailability of organic phosphorus in the water level fluctuation zone soil and the effects of ultraviolet irradiation on it in the Three Gorges Reservoir, China. Sci. Total Environ.; 2020; 738, 139912. [DOI: https://dx.doi.org/10.1016/j.scitotenv.2020.139912] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32531607]

12. Wang, C.; Guo, J.S.; Zhang, W.; Jiang, Y.X.; Fang, F.; He, W.Z.; Jia, B.Y.; Dang, C.J. Drying-rewetting changes soil phosphorus status and enzymatically hydrolysable organic phosphorus fractions in the water-level fluctuation zone of Three Gorges reservoir. Catena; 2021; 204, 105416. [DOI: https://dx.doi.org/10.1016/j.catena.2021.105416]

13. Jarosch, K.A.; Kandeler, E.; Frossard, E.; Bunemann, E.K. Is the enzymatic hydrolysis of soil organic phosphorus compounds limited by enzyme or substrate availability?. Soil Biol. Biochem.; 2019; 139, 107628. [DOI: https://dx.doi.org/10.1016/j.soilbio.2019.107628]

14. Wei, K.; Sun, T.; Tian, J.H.; Chen, Z.H.; Chen, L.J. Soil microbial biomass, phosphatase and their relationships with phosphorus turnover under mixed inorganic and organic nitrogen addition in a Larix gmelinii plantation. Forest Ecol. Manag.; 2018; 422, pp. 313-322. [DOI: https://dx.doi.org/10.1016/j.foreco.2018.04.035]

15. Zhu, J.; Qu, B.; Li, M. Phosphorus mobilization in the Yeyahu Wetland: Phosphatase enzyme activities and organic phosphorus fractions in the rhizosphere soils. Int. Biodeter. Biodegr.; 2017; 124, pp. 304-313. [DOI: https://dx.doi.org/10.1016/j.ibiod.2017.05.010]

16. Nakayama, Y.; Wade, J.; Margenot, A.J. Does soil phosphomonoesterase activity reflect phosphorus pools estimated by Hedley phosphorus fractionation?. Geoderma; 2021; 401, 115279. [DOI: https://dx.doi.org/10.1016/j.geoderma.2021.115279]

17. Tuszynska, A.; Czerwionka, K.; Obarska-Pempkowiak, H. Phosphorus concentration and availability in raw organic waste and post fermentation products. J. Environ. Manag.; 2021; 278, 111468. [DOI: https://dx.doi.org/10.1016/j.jenvman.2020.111468]

18. Zhang, Z.Y.; Hu, H.Q.; Wan, C.Y.; Peng, J.H.; Xu, F.L.; Shi, F. Lateral and longitudinal variation in phosphorus fractions in surface sediment and adjacent riparian soil in the Three Gorges Reservoir, China. Environ. Sci. Pollut. R; 2018; 25, pp. 31262-31271. [DOI: https://dx.doi.org/10.1007/s11356-018-3087-7]

19. Blomback, K.; Bolster, C.H.; Lindsjo, A.; Hesse, K.; Linefur, H.; Parvage, M.M. Comparing measures for determination of phosphorus saturation as a method to estimate dissolved P in soil solution. Geoderma; 2021; 383, 114708. [DOI: https://dx.doi.org/10.1016/j.geoderma.2020.114708]

20. Wu, A.; Fang, Y.; Liu, S.; Wang, H.; Xu, B.; Zhang, S.; Deng, X.; Palta, J.A.; Siddique, K.H.M.; Chen, Y. Root morphology and rhizosheath acid phosphatase activity in legume and graminoid species respond differently to low phosphorus supply. Rhizosphere; 2021; 19, 100391. [DOI: https://dx.doi.org/10.1016/j.rhisph.2021.100391]

21. Luo, G.W.; Sun, B.; Li, L.; Li, M.H.; Liu, M.Q.; Zhu, Y.Y.; Guo, S.W.; Ling, N.; Shen, Q.R. Understanding how long-term organic amendments increase soil phosphatase activities: Insight into phoD- and phoC-harboring functional microbial populations. Soil Biol. Biochem.; 2019; 139, 107632. [DOI: https://dx.doi.org/10.1016/j.soilbio.2019.107632]

22. Bing, H.J.; Zhou, J.; Wu, Y.H.; Wang, X.X.; Sun, H.Y.; Li, R. Current state, sources, and potential risk of heavy metals in sediments of Three Gorges Reservoir, China. Environ. Pollut.; 2016; 214, pp. 485-496. [DOI: https://dx.doi.org/10.1016/j.envpol.2016.04.062] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27131806]

23. Yue, J.S.; Yuan, X.Z.; Li, B.; Ren, H.Q.; Wang, X.F. Emergy and exergy evaluation of a dike-pond project in the drawdown zone (DDZ) of the Three Gorges Reservoir (TGR). Ecol. Indic.; 2016; 71, pp. 248-257. [DOI: https://dx.doi.org/10.1016/j.ecolind.2016.05.015]

24. Cui, Y.X.; Bing, H.J.; Fang, L.C.; Jiang, M.; Shen, G.T.; Yu, J.L.; Wang, X.; Zhu, H.; Wu, Y.H.; Zhang, X.C. Extracellular enzyme stoichiometry reveals the carbon and phosphorus limitations of microbial metabolisms in the rhizosphere and bulk soils in alpine ecosystems. Plant Soil; 2021; 458, pp. 7-20. [DOI: https://dx.doi.org/10.1007/s11104-019-04159-x]

25. Zhang, X.; Zhang, R.; Gao, J.; Wang, X.; Fan, F.; Ma, X.; Yin, H.; Zhang, C.; Feng, K.; Deng, Y. Thirty-one years of rice-rice-green manure rotations shape the rhizosphere microbial community and enrich beneficial bacteria. Soil Biol. Biochem.; 2017; 104, pp. 208-217. [DOI: https://dx.doi.org/10.1016/j.soilbio.2016.10.023]

26. Salehi, M.H.; Beni, O.H.; Harchegani, H.B.; Borujeni, I.E.; Motaghian, H.R. Refining Soil Organic Matter Determination by Loss-on-Ignition. Pedosphere; 2011; 21, pp. 473-482. [DOI: https://dx.doi.org/10.1016/S1002-0160(11)60149-5]

27. Brookes, P.C.; Powlson, D.S.; Jenkinson, D.S. Measurement of microbial biomass phosphorus in soil. Soil Biol. Biochem.; 1982; 14, pp. 319-329. [DOI: https://dx.doi.org/10.1016/0038-0717(82)90001-3]

28. Ruban, V.; Lopez-Sanchez, J.F.; Pardo, P.; Rauret, G.; Muntau, H.; Quevauviller, P. Selection and evaluation of sequential extraction procedures for the determination of phosphorus forms in lake sediment. J. Environ. Monit.; 1999; 1, pp. 51-56. [DOI: https://dx.doi.org/10.1039/a807778i] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/11529079]

29. Murphy, J.; Riley, J.P. A modified single solution method for the determination of phosphate in natural-waters. Anal. Chim. Acta; 1962; 27, pp. 31-36. [DOI: https://dx.doi.org/10.1016/S0003-2670(00)88444-5]

30. Tiessen, H.; Moir, J.O. Characterization of available P by sequential extraction. Soil Sampling and Methods of Analysis; 2nd ed. Carter, M.R. Lewis Publishers: Ann Arbor, MI, USA, 1993; pp. 75-86.

31. Sun, W.G.; Villamil, M.B.; Behnke, G.D.; Margenot, A.J. Long-term effects of crop rotation and nitrogen fertilization on phosphorus cycling and balances in loess-derived Mollisols. Geoderma; 2022; 420, 115829. [DOI: https://dx.doi.org/10.1016/j.geoderma.2022.115829]

32. Hamilton, G.A. Inositol phosphates, their chemistry, biochemistry and physiology-cosgrove,dj. J. Am. Chem. Soc.; 1983; 105, 2515.

33. Gu, C.; Wilson, S.G.; Margenot, A.J. Lithological and bioclimatic impacts on soil phosphatase activities in California temperate forests. Soil Biol. Biochem.; 2020; 141, 107633. [DOI: https://dx.doi.org/10.1016/j.soilbio.2019.107633]

34. Tibbett, M. Considerations on the use of the p-nitrophenyl phosphomonoesterase assay in the study of the phosphorus nutrition of soil borne fungi. Microbiol. Res.; 2002; 157, pp. 221-231. [DOI: https://dx.doi.org/10.1078/0944-5013-00154] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/12398293]

35. Margenot, A.J.; Nakayama, Y.; Parikh, S.J. Methodological recommendations for optimizing assays of enzyme activities in soil samples. Soil Biol. Biochem.; 2018; 125, pp. 350-360. [DOI: https://dx.doi.org/10.1016/j.soilbio.2017.11.006]

36. Tamaru, S.; Yabuta, S.; Goto, K.; Khanthavong, P.; Sakagami, J.I. Oxygen consumption and transportation in rice roots are highly dependent on leaf area and root dry weight ratio. Rhizosphere; 2023; 25, 100644. [DOI: https://dx.doi.org/10.1016/j.rhisph.2022.100644]

37. Hu, L.Y.; Li, D.; Sun, K.; Cao, W.; Fu, W.Q.; Zhang, W.; Dai, C.C. Mutualistic fungus Phomopsis liquidambari increases root aerenchyma formation through auxin-mediated ethylene accumulation in rice (Oryza sativa L.). Plant Physiol. Biochem.; 2018; 130, pp. 367-376. [DOI: https://dx.doi.org/10.1016/j.plaphy.2018.07.018]

38. Dittrich, M.; Chesnyuk, A.; Gudimo, A.; McCulloch, J.; Quazi, S.; Young, J.; Winter, J.; Stainsby, E.; Arhonditsis, G. Phosphorus retention in a mesotrophic lake under transient loading conditions: Insights from a sediment phosphorus binding form study. Water Res.; 2013; 47, pp. 1433-1447. [DOI: https://dx.doi.org/10.1016/j.watres.2012.12.006]

39. Zhang, Y.; Xu, J.; Riera, N.; Jin, T.; Li, J.; Wang, N. Huanglongbing impairs the rhizosphere-to-rhizoplane enrichment process of the citrus root-associated microbiome. Microbiome; 2017; 5, 97. [DOI: https://dx.doi.org/10.1186/s40168-017-0304-4]

40. Singh, J.P.; Ojinnaka, E.U.; Krumins, J.A.; Goodey, N.M. Abiotic factors determine functional outcomes of microbial inoculation of soils from a metal contaminated brownfield. Ecotoxicol. Environ. Saf.; 2019; 168, pp. 450-456. [DOI: https://dx.doi.org/10.1016/j.ecoenv.2018.10.114]

41. Bargaz, A.; Elhaissoufi, W.; Khourchi, S.; Benmrid, B.; Borden, K.A.; Rchiad, Z. Benefits of phosphate solubilizing bacteria on belowground crop performance for improved crop acquisition of phosphorus. Microbiol. Res.; 2021; 252, 126842. [DOI: https://dx.doi.org/10.1016/j.micres.2021.126842]

42. Grigatti, M.; Boanini, E.; Mancarella, S.; Simoni, A.; Centemero, M.; Veeken, A.H.M. Phosphorous extractability and ryegrass availability from bio-waste composts in a calcareous soil. Chemosphere; 2017; 174, pp. 722-731. [DOI: https://dx.doi.org/10.1016/j.chemosphere.2017.02.039]

43. Qu, Y.; Wang, C.; Guo, J.S.; Huang, J.J.; Fang, F.; Xiao, Y.; Ouyang, W.J.; Lu, L.H. Characteristics of organic phosphorus fractions in soil from water-level fluctuation zone by solution P-31-nuclear magnetic resonance and enzymatic hydrolysis. Environ. Pollut.; 2019; 255, 113209. [DOI: https://dx.doi.org/10.1016/j.envpol.2019.113209]

44. Chen, Z.L.; Fang, F.; Shao, Y.; Jiang, Y.X.; Huang, J.J.; Guo, J.S. The biotransformation of soil phosphorus in the water level fluctuation zone could increase eutrophication in reservoirs. Sci. Total Environ.; 2021; 763, 142976. [DOI: https://dx.doi.org/10.1016/j.scitotenv.2020.142976]

45. Fujii, K.; Funakawa, S.; Kosaki, T. Effects of forest management on soil acidification in cedar plantation. Geoderma; 2022; 424, 115967. [DOI: https://dx.doi.org/10.1016/j.geoderma.2022.115967]

46. Li, C.; Ding, S.M.; Chen, M.S.; Sun, Q.; Zhang, Y.; Ma, X.; Zhong, Z.L.; Tsang, D.; Wang, Y. Mechanistic insights into trace metal mobilization at the micro-scale in the rhizosphere of Vallisneria spiralis. Sci. Total Environ.; 2022; 806, 150735. [DOI: https://dx.doi.org/10.1016/j.scitotenv.2021.150735]

47. Audette, Y.; O'Halloran, I.P.; Evans, L.J.; Martin, R.C.; Voroney, R.P. Kinetics of phosphorus forms applied as inorganic and organic amendments to a calcareous soil II: Effects of plant growth on plant available and uptake phosphorus. Geoderma; 2016; 279, pp. 70-76. [DOI: https://dx.doi.org/10.1016/j.geoderma.2016.06.002]

48. Sun, B.R.; Gao, Y.Z.; Wu, X.; Ma, H.M.; Zheng, C.C.; Wang, X.Y.; Zhang, H.L.; Li, Z.J.; Yang, H.J. The relative contributions of pH, organic anions, and phosphatase to rhizosphere soil phosphorus mobilization and crop phosphorus uptake in maize/alfalfa polyculture. Plant Soil; 2020; 447, pp. 117-133. [DOI: https://dx.doi.org/10.1007/s11104-019-04110-0]

49. Hu, Y.W.; Duan, C.Q.; Fu, D.G.; Wu, X.I.; Yan, K.; Fernando, E.; Karunarathna, S.C.; Promputtha, I.; Mortimer, P.E.; Xu, J.C. Structure of bacterial communities in phosphorus-enriched rhizosphere soils. Appl. Sci.; 2020; 10, 6387. [DOI: https://dx.doi.org/10.3390/app10186387]

50. Fu, D.G.; Wu, X.N.; Duan, C.Q.; Zhao, L.Q.; Li, B. Different life-form plants exert different rhizosphere effects on phosphorus biogeochemistry in subtropical mountainous soils with low and high phosphorus content. Soil Till. Res.; 2020; 199, 104516. [DOI: https://dx.doi.org/10.1016/j.still.2019.104516]

51. Almeida, D.S.; Penn, C.J.; Rosolem, C.A. Assessment of phosphorus availability in soil cultivated with ruzigrass. Geoderma; 2018; 312, pp. 64-73. [DOI: https://dx.doi.org/10.1016/j.geoderma.2017.10.003]

52. Hazarika, S.; Sohliya, B.; Thakuria, D.; Kataki, S.; Rangappa, K. Influence of organic amendments on acidic soil responsive crop groundnut (Arachis hypogaea L.). Environ. Prog. Sustain.; 2021; 40, 13592. [DOI: https://dx.doi.org/10.1002/ep.13592]

53. Hoang, D.; Rashtbari, M.; Anh, L.T.; Wang, S.; Tu, D.T.; Hiep, N.V.; Razavi, B.S. Mutualistic interaction between arbuscular mycorrhiza fungi and soybean roots enhances drought resistant through regulating glucose exudation and rhizosphere expansion. Soil Biol. Biochem.; 2022; 171, 108728. [DOI: https://dx.doi.org/10.1016/j.soilbio.2022.108728]

54. Richardson, A.E.; Hocking, P.J.; Simpson, R.J.; George, T.S. Plant mechanisms to optimise access to soil phosphorus. Crop. Pasture Sci.; 2009; 60, pp. 124-143. [DOI: https://dx.doi.org/10.1071/CP07125]

55. Meier, S.; de Souza Campos, P.; Palma-Millanao, R.; Morales, A.; Hirzel, J.; Aponte, H.; Cartes, P.; Ondrasek, G.; Seguel, A. Assembly between wheat cultivars and soil microorganisms modulates phosphorus and water use efficiency. Rhizosphere; 2023; 25, 100631. [DOI: https://dx.doi.org/10.1016/j.rhisph.2022.100631]

56. Pii, Y.; Mimmo, T.; Tomasi, N.; Terzano, R.; Cesco, S.; Crecchio, C. Microbial interactions in the rhizosphere: Beneficial influences of plant growth-promoting rhizobacteria on nutrient acquisition process. A review. Biol. Fert. Soils; 2015; 51, pp. 403-415. [DOI: https://dx.doi.org/10.1007/s00374-015-0996-1]

57. Holz, M.; Zarebanadkouki, M.; Carminati, A.; Becker, J.N.; Spohn, M. The effect of root hairs on rhizosphere phosphatase activity. J. Plant Nutr. Soil Sci.; 2020; 183, pp. 382-388. [DOI: https://dx.doi.org/10.1002/jpln.201900426]

58. Tian, P.; Razavi, B.S.; Zhang, X.C.; Wang, Q.K.; Blagodatskaya, E. Microbial growth and enzyme kinetics in rhizosphere hotspots are modulated by soil organics and nutrient availability. Soil Biol. Biochem.; 2020; 141, 107662. [DOI: https://dx.doi.org/10.1016/j.soilbio.2019.107662]

59. Zhu, Y.R.; Wu, F.C.; He, Z.Q.; Guo, J.Y.; Qu, X.X.; Xie, F.Z.; Giesy, J.P.; Liao, H.Q.; Guo, F. Characterization of organic phosphorus in lake sediments by sequential fractionation and enzymatic hydrolysis. Environ. Sci. Technol.; 2013; 47, pp. 7679-7687. [DOI: https://dx.doi.org/10.1021/es305277g]

60. Lai, Y.C.; Syu, C.H.; Wang, P.J.; Lee, D.Y.; Fan, C.; Juang, K.W. Field experiment for determining lead accumulation in rice grains of different genotypes and correlation with iron oxides deposited on rhizosphere soil. Sci. Total Environ.; 2018; 610, pp. 845-853. [DOI: https://dx.doi.org/10.1016/j.scitotenv.2017.08.034] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28826122]

61. Margalef, O.; Sardans, J.; Fernandez-Martinez, M.; Molowny-Horas, R.; Janssens, I.A.; Ciais, P.; Goll, D.; Richter, A.; Obersteiner, M.; Asensio, D. et al. Global patterns of phosphatase activity in natural soils. Sci. Rep.; 2017; 7, 1337. [DOI: https://dx.doi.org/10.1038/s41598-017-01418-8]

62. Olsson, R.; Giesler, R.; Loring, J.S.; Persson, P. Enzymatic Hydrolysis of Organic Phosphates Adsorbed on Mineral Surfaces. Environ. Sci. Technol.; 2012; 46, pp. 285-291. [DOI: https://dx.doi.org/10.1021/es2028422] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/22103404]

63. Yang, M.; Lu, Y.; Mu, L.; Yang, H. Leaf and soil phosphatases and the correlations with leaf P resorption of lucerne under P fertilization and irrigation. J. Soil Sci. Plant Nutr.; 2023; 23, pp. 842-853. [DOI: https://dx.doi.org/10.1007/s42729-022-01087-1]

64. Fischer, P.; Pothig, R.; Venohr, M. The degree of phosphorus saturation of agricultural soils in Germany: Current and future risk of diffuse P loss and implications for soil P management in Europe. Sci. Total Environ.; 2017; 599, pp. 1130-1139. [DOI: https://dx.doi.org/10.1016/j.scitotenv.2017.03.143] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28511358]

65. Pothig, R.; Behrendt, H.; Opitz, D.; Furrer, G. A universal method to assess the potential of phosphorus loss from soil to aquatic ecosystems. Environ. Sci. Pollut. R; 2010; 17, pp. 497-504. [DOI: https://dx.doi.org/10.1007/s11356-009-0230-5] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/19705176]

66. Tang, Y.Y.; Van Kempen, M.; Van der Heide, T.; Manschot, J.; Roelofs, J.; Lamers, L.; Smolders, A. A tool for easily predicting short-term phosphorus mobilization from flooded soils. Ecol. Eng.; 2016; 94, pp. 1-6. [DOI: https://dx.doi.org/10.1016/j.ecoleng.2016.05.046]

67. Chen, X.M.; Zhang, W.; Gruau, G.; Couic, E.; Cotinet, P.; Li, Q.M. Conservation practices modify soil phosphorus sorption properties and the composition of dissolved phosphorus losses during runoff. Soil Till. Res.; 2022; 220, 105353. [DOI: https://dx.doi.org/10.1016/j.still.2022.105353]

68. Steinfurth, K.; Borjesson, G.; Denoroy, P.; Eichler-Lobermann, B.; Gans, W.; Heyn, J.; Hirte, J.; Huyghebaert, B.; Jouany, C.; Koch, D. et al. Thresholds of target phosphorus fertility classes in European fertilizer recommendations in relation to critical soil test phosphorus values derived from the analysis of 55 European long-term field experiments. Agric. Ecosyst. Environ.; 2022; 332, 107926. [DOI: https://dx.doi.org/10.1016/j.agee.2022.107926]

69. Qu, H.Y.; Jia, G.M.; Liu, X.; Xiang, H.Y.; Yue, Y.F.; Zhang, H.L. Soil Extracellular Enzymatic Stoichiometry along a Hydrologic Gradient of Hillslope Riparian Zone of the Three Gorges Reservoir. Soil Sci. Soc. Am. J.; 2019; 83, pp. 1575-1584. [DOI: https://dx.doi.org/10.2136/sssaj2018.12.0509]

70. Browman, M.G.; Tabatabai, M.A. Phosphodiesterase Activity of Soils. Soil Sci. Soc. Am. J.; 1978; 42, pp. 284-290. [DOI: https://dx.doi.org/10.2136/sssaj1978.03615995004200020016x]

71. McLachlan, K.D. Acid-phosphatase-activity of intact roots and phosphorus-nutrition in plants. 1. Assay conditions and phosphatase-activity. Aus. J. Agric. Res.; 1980; 31, pp. 429-440. [DOI: https://dx.doi.org/10.1071/AR9800429]

72. Tabatabai, M.A.; Bremner, J.M. Use of p-nitrophenyl phosphate for assay of soil phosphatase activity. Soil Biol. Biochem.; 1969; 1, pp. 301-307. [DOI: https://dx.doi.org/10.1016/0038-0717(69)90012-1]

73. Turner, B.L.; Haygarth, P.M. Phosphatase activity in temperate pasture soils: Potential regulation of labile organic phosphorus turnover by phosphodiesterase activity. Sci. Total Environ.; 2005; 344, pp. 27-36. [DOI: https://dx.doi.org/10.1016/j.scitotenv.2005.02.003] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15907508]