Exogenous Glomalin-Related Soil Proteins Differentially Regulate Soil Properties in Trifoliate Orange

: Glomalin-related soil protein (GRSP) is a speciﬁc glycoprotein secreted into the soil by hyphae and spores of arbuscular mycorrhizal fungi that have many potential functions. It is not clear whether exogenous GRSP has an effect on plant growth and soil properties or whether the effects are related to the type of GRSP used. In this study, trifoliate orange ( Poncirus trifoliata L. Raf.) seedlings were used to analyze the effects of easily extractable GRSP (EE-GRSP) and difﬁcultly extractable GRSP (DE-GRSP) at a quarter-, half-, and full-strength concentration on shoot and root biomass as well as soil properties The results showed that, at different strengths, exogenous EE-GRSR signiﬁcantly increased shoot and root biomass compared to the control, which displayed the most signiﬁcant effects from the half-strength EE-GRSP. In contrast, DE-GRSP, at various strengths, signiﬁcantly reduced shoot and root biomass. Furthermore, the application of exogenous EE-GRSP stimulated soil water-stable aggregate (WSA) content at 2–4 mm and 0.5–1 mm sizes, while DE-GRSP strongly reduced WSA content at the 2–4 mm, 1–2 mm, 0.5–1 mm, and 0.25–0.5 mm sizes, consequently leading to an increase or decrease in the WSA stability, according to the mean weight diameter. However, exogenous EE-GRSP decreased soil pH and DE-GRSP increased it, which was related to WSA stability. Exogenous EE-GRSP signiﬁcantly increased soil acidic, neutral, and alkaline phosphatase activity at different strengths, while exogenous DE-GRSP, also at different strengths, signiﬁcantly inhibited soil acidic phosphatase activity. The application of both exogenous EE-GRSP and DE-GRSP increased the organic carbon content of the soil. This study concluded that exogenous GRSP exerted differential effects on plant biomass and soil properties, and EE-GRSP can be considered as a soil stimulant for use in citrus plants. To our knowledge, this is the ﬁrst report on the negative effects of exogenous DE-GRSP on plant biomass and soil properties. soil properties. Furthermore, we also observed that exogenous DE-GRSP exhibited adverse effects on plant biomass, WSA formation and stability, soil pH, and soil acid phosphatase activity. Such results guide the management of soil GRSP in citrus orchards for reducing DE-GRSP and EE-GRSP.


Introduction
Arbuscular mycorrhizal fungi (AMF) form symbiotic relationships with approximately 80% of terrestrial plants. After colonizing the roots of host plants, AMF develop mycorrhizal extraradical hyphae, which extend into the soil beyond the root, thus increasing the root surface area of the plant and its contact with the soil/rhizosphere [1,2]. As a result, arbuscular mycorrhizas promote the growth and development of the host plant by improving the fruit quality, water, and nutrient acquisition, in addition to increasing its stress resistance [2][3][4][5][6][7]. The beneficial effects of mycorrhizal symbiosis are partly attributed to the production of a specific glycoprotein, glomalin, which is secreted by the hyphae and spores of AMF [8][9][10]. Rillig [11] proposed the term glomalin-related soil protein (GRSP) to describe the soil with glomalin, based on the Bradford [12] assay. For the extraction of GRSP, Wu et al. [13] divided GRSP into easily extractable GRSP (EE-GRSP) and the more difficult to extract GRSP (DE-GRSP). GRSP can attach to the outside of soil aggregates and reduce the loss of soil, water, and nutrients [14]. In addition, glomalin can enter the soil with the degradation of extraradical mycelium, which in turn plays a very important role in improving the organic carbon sequestration and soil properties [15]. It is documented that GRSP promotes the formation and stability of soil aggregates, in addition to improving soil and plant water relations [14,16].
GRSP is considered to be insoluble in water and difficult to decompose in its natural state; therefore, it is extremely stable with a turnover time of 6-42 years [17,18]. GRSP is mainly composed of protein bodies, carbohydrates, iron, and other ions; the iron content reaches 1%-9%, and the type and content of cations in GRSP vary greatly depending upon the nature of the soil [19]. Gadkar and Rillig [20] found that the sequence of GRSP was highly homologous to heat shock protein 60 (Hsp6o) with >80% homology. This was found by sequencing with liquid chromatography-mass spectrometry.
In GRSP fractions, EE-GRSP is considered to be newly generated by AMF and relatively more labile [21]. Studies have attempted to use EE-GRSP as a plant/soil stimulant. It was demonstrated that exogenous EE-GRSP increased the organic carbon content and phosphatase activity of soil in citrus orchards [22] as well as the stability of soil aggregates and the content of water-stable aggregates [21]. However, the other GRSP fraction, DE-GRSP, originates from the turnover of EE-GRSP and thus an older glomalin [23]. To date, there remains no information regarding the effects of exogenous DE-GRSP application on plant growth and soil properties. Therefore, the aim of this study is to apply EE-GRSP and DE-GRSP at different concentrations on trifoliate orange seedlings and evaluate the effects on biomass production, soil aggregate stability, SOC, phosphatase, and pH value.

Plant Culture
The seed of trifoliate orange (Poncirus trifoliata L. Raf.) was soaked in 1 mol/L sodium hydroxide solution for 10 min, disinfected with 75% alcohol for 10 min, and washed with distilled water for 4 times. Subsequently, the seeds were placed in sterilized river sand (φ ≤ 2 mm) and incubated at 28 • C/20 • C (day/night) with 68% relative air humidity. A month later, the three seedlings with four leaves were transplanted into a 2.1 L plastic pot with a 4 mm diameter that was pre-loaded with soil and sterilized (0.11 MPa, 121 • C) for 2 h. The physicochemical characteristics of the soil are shown in Table 1. The plants were placed in a greenhouse, where the light intensity was 900 µmol/m 2 /s, the temperature was 28 • C/20 • C (day/night), and the relative humidity was 68%.

Preparation and Application of GRSP and Experimental Design
The soil from the citrus orchard in Yangtze University was collected, air-dried, and sieved to remove stones and root segments, and then EE-GRSP and DE-GRSP were extracted by the method of Wu et al. [23]. A 0.5 g soil sample was mixed with 4 mL of 20 mmol/L citrate buffer (pH 7.0), extracted at 103 kPa, heated to 121 • C for 30 min, and centrifuged at 10,000× g/min for 5 min. The supernatant was intended to be the fullstrength EE-GRSP (1 GRSP). The centrifugal residue continued to be mixed with 4 mL of 50 mmol/L sodium citrate buffer (pH 8), extracted at 103 kPa and heated to 121 • C for 60 min, and centrifuged at 10,000× g/min for 10 min. This supernatant was selected as full-strength DE-GRSP. The protein concentration of full-strength EE-GRSP and DE-GRSP was 0.24 mg/L and 0.36 mg/L, respectively, according to Bradford's [12] method, with bovine serum protein as the standard.
Different strengths of EE-GRSP and DE-GRSP were prepared with dilutions of fullstrength GRSP with 20 mmol/L citrate buffers (pH 7.0) and 50 mmol/L sodium citrate buffers (pH 8), respectively. After two weeks of seedling transplanting, 50 mL of different concentrations of GRSP was applied to the pot weekly, and the same amount of citrate buffer (pH 7.0) was applied to the control group. The experiment ended after 16 weeks of exogenous GRSP application.
In this experiment, a completely randomized block design was carried out, in which seven treatments, namely, the control (0 GRSP), quarter-strength EE-GRSP, half-strength EE-GRSP, full-strength EE-GRSP, quarter-strength DE-GRSP, half-strength DE-GRSP, and full-strength DE-GRSP, were arranged. Each treatment had four replicates, with two pots for each replicate, totaling 56 pots. The pots were arranged randomly, and their positions were changed regularly to decrease the impact of light on pot locations.

Variable Determinations
Plants were separated from the shoot and root parts at harvest, and then their fresh weight was measured. At harvest time, the soil sticking to the root surface was gently shaken off, air-dried, and collected for the analysis of particular soil properties.
Soil pH value was determined as per the protocol outlined by Cheng et al. [24] with a pH meter (PH828+, Smart Sensor, Arco Science & Technology Ltd., Dongguan, China).
Soil water-stable aggregates (WSAs) in the sizes of 2-4 mm, 1-2 mm, 0.5-1 mm, and 0.25-0.5 mm were assayed according to the wet-sieving method [25]. Soil WSA stability was measured according to the analysis of the mean weight diameter (MWD) with the following formula [25]: where X i is the mean diameter of the i sieve opening (mm), W i is the proportion of the i size fraction in the total sample mass, and n is number of size fractions. SOC was determined by the dichromate oxidation spectrophotometric method as outlined by Rowell [26]. Determination of soil acid phosphatase (ACP, extracted by 0.1 mmol/L sodium acetate buffer at pH 5.0), neutral phosphatase (NEP, extracted by 20 mmol/L citrate buffer at pH 7.0), and alkaline phosphatase (ALP, extracted by 0.2 mmol/L borate buffer at pH 10.0) activity was assayed by the method of Zhao and Jiang [27], using disodium phenyl phosphate as the substrate and redistilled phenol as the standard.
The extraction and analysis of EE-GRSP and DE-GRSP in soil was previously described in Section 2.2.

Statistical Analysis
The one-way analysis of variance (ANOVA) in SAS software (v8.1) was used to analyze the experimental data, and Duncan's multiple-range test was used to compare significant differences among treatments (p < 0.05). Figures were generated using SigmaPlot (v10.0).

Changes in Biomass Production
After treatment with exogenous EE-GRSP, the EE-GRSP-treated plants were recorded as having significantly higher shoot and root biomass of trifoliate orange seedlings. A total of 39% and 19% of the shoots and roots received a quarter-strength of EE-GRSP application, 52% and 53% received a half-strength EE-GRSP application, and 49% and 34% received a full-strength EE-GRSP application, respectively ( Figure 1a). Evidently, the half-strength EE-GRSP treatment provided the best promotive effects. However, compared with the control (0 GRSP), all DE-GRSP treatments significantly reduced shoot and root biomass production with the increase in DE-GRSP concentrations (Figure 1b). Among them, the quarter-strength DE-GRSP considerably reduced shoot and root biomass by 51% and 53%, the half-strength DE-GRSP by 56% and 56%, and the full-strength DE-GRSP by 71% and 72%, respectively. application, 52% and 53% received a half-strength EE-GRSP application, and 49% and 34% received a full-strength EE-GRSP application, respectively ( Figure 1a). Evidently, the halfstrength EE-GRSP treatment provided the best promotive effects. However, compared with the control (0 GRSP), all DE-GRSP treatments significantly reduced shoot and root biomass production with the increase in DE-GRSP concentrations (Figure 1b). Among them, the quarter-strength DE-GRSP considerably reduced shoot and root biomass by 51% and 53%, the half-strength DE-GRSP by 56% and 56%, and the full-strength DE-GRSP by 71% and 72%, respectively.

Figure 1.
Effect of exogenous EE-GRSP and DE-GRSP on shoot (a) and root (b) biomass production of trifoliate orange seedlings. Data (mean ± SD, n = 4) followed by different letters above the bars indicate significant differences (p < 0.05) between treatments.

Changes in Soil WSA Distribution and Stability
Exogenous GRSP treatment strongly affected the distribution of WSAs in the rhizosphere of trifoliate orange seedlings ( Table 2). All EE-GRSP treatments significantly increased WSA percentages in the 2-4 mm and 0.5-1 mm sizes compared with the control (0 GRSP). In addition, the quarter-and full-strength EE-GRSP also significantly increased WSA percentages in the 1-2 mm size, and the half-strength EE-GRSP significantly increased WSA percentages in the 0.25-0.5 mm size. Three DE-GRSP treatments collectively reduced WSA percentages in the 2-4 mm, 1-2 mm, 0.5-1 mm, and 0.25-0.5 mm sizes. In addition, compared to the control, the quarter-, half-, and full-strength EE-GRSP significantly increased MWD by 19%, 20%, and 16%, respectively; quarter-, half-, and fullstrength DE-GRSP significantly decreased MWD by 55%, 61%, and 65%, respectively.

Changes in Soil WSA Distribution and Stability
Exogenous GRSP treatment strongly affected the distribution of WSAs in the rhizosphere of trifoliate orange seedlings ( Table 2). All EE-GRSP treatments significantly increased WSA percentages in the 2-4 mm and 0.5-1 mm sizes compared with the control (0 GRSP). In addition, the quarter-and full-strength EE-GRSP also significantly increased WSA percentages in the 1-2 mm size, and the half-strength EE-GRSP significantly increased WSA percentages in the 0.25-0.5 mm size. Three DE-GRSP treatments collectively reduced WSA percentages in the 2-4 mm, 1-2 mm, 0.5-1 mm, and 0.25-0.5 mm sizes. In addition, compared to the control, the quarter-, half-, and full-strength EE-GRSP significantly increased MWD by 19%, 20%, and 16%, respectively; quarter-, half-, and full-strength DE-GRSP significantly decreased MWD by 55%, 61%, and 65%, respectively. Data (mean ± SD, n =4) followed by the different letters among treatments indicate significant differences at p < 0.05.
Regression analysis showed a significant curvilinear correlation between MWD and exogenous EE-GRSP concentrations, with a trend of increasing and then decreasing with a peak between 0.006-0.012 mg of protein/mL citrate buffer (Figure 2a). In addition, MWD values showed a significant curvilinear correlation in the concentration of DE-GRSP (Figure 2b), with a trend of decreasing and then slightly increasing to reach a trough between 0.009-0.018 mg of protein/mL citrate buffer. exogenous EE-GRSP concentrations, with a trend of increasing and then decreasing with a peak between 0.006-0.012 mg of protein/mL citrate buffer (Figure 2a). In addition, MWD values showed a significant curvilinear correlation in the concentration of DE-GRSP (Figure 2b), with a trend of decreasing and then slightly increasing to reach a trough between 0.009-0.018 mg of protein/mL citrate buffer.

Changes in Soil pH Value
The application of exogenous GRSP significantly changed the soil pH value. The reduction in the soil pH value was caused by exogenous EE-GRSP and the increase by exogenous DE-GRSP (Figure 3). There were no significant differences in soil pH value among the three EE-GRSP treatments or the three DE-GRSP treatments. In addition, the soil pH value was substantially and negatively correlated with MWD ( Figure 4).

Changes in Soil pH Value
The application of exogenous GRSP significantly changed the soil pH value. The reduction in the soil pH value was caused by exogenous EE-GRSP and the increase by exogenous DE-GRSP ( Figure 3). There were no significant differences in soil pH value among the three EE-GRSP treatments or the three DE-GRSP treatments. In addition, the soil pH value was substantially and negatively correlated with MWD ( Figure 4).

Changes in SOC Content
Exogenous EE-GRSP and DE-GRSP application considerably increased the SOC content ( Figure 5). Among the treatments, the SOC quantity rose with the increase in exogenous DE-GRSP concentration. Between the three EE-GRSP treatments, the half-strength EE-GRSP application showed a relatively better effect of increasing the SOC. A significantly and positively linear relationship was observed between the SOC and the exogenous EE-GRSP concentration (Figure 6a) and the DE-GRSP concentration (Figure 6b), respectively. Data (mean ± SD, n =4) followed by the different letters among treatments indicate significant differences at p < 0.05.
Regression analysis showed a significant curvilinear correlation between MWD and exogenous EE-GRSP concentrations, with a trend of increasing and then decreasing with a peak between 0.006-0.012 mg of protein/mL citrate buffer (Figure 2a). In addition, MWD values showed a significant curvilinear correlation in the concentration of DE-GRSP (Figure 2b), with a trend of decreasing and then slightly increasing to reach a trough between 0.009-0.018 mg of protein/mL citrate buffer.

Changes in Soil pH Value
The application of exogenous GRSP significantly changed the soil pH value. The reduction in the soil pH value was caused by exogenous EE-GRSP and the increase by exogenous DE-GRSP ( Figure 3). There were no significant differences in soil pH value among the three EE-GRSP treatments or the three DE-GRSP treatments. In addition, the soil pH value was substantially and negatively correlated with MWD ( Figure 4).  Effects of exogenous EE-GRSP and DE-GRSP on the soil pH value in the rhizosphere of trifoliate orange seedlings. Data (means ± SD, n = 4) followed by different letters above the bars indicate significant differences (p < 0.05) between treatments.

Changes in SOC Content
Exogenous EE-GRSP and DE-GRSP application considerably increased the SOC content ( Figure 5). Among the treatments, the SOC quantity rose with the increase in exogenous DE-GRSP concentration. Between the three EE-GRSP treatments, the half-strength EE-GRSP application showed a relatively better effect of increasing the SOC. A significantly and positively linear relationship was observed between the SOC and the exogenous EE-GRSP concentration (Figure 6a) and the DE-GRSP concentration (Figure 6b), respectively.

Changes in SOC Content
Exogenous EE-GRSP and DE-GRSP application considerably increased the SOC content ( Figure 5). Among the treatments, the SOC quantity rose with the increase in exogenous DE-GRSP concentration. Between the three EE-GRSP treatments, the half-strength EE-GRSP application showed a relatively better effect of increasing the SOC. A significantly and positively linear relationship was observed between the SOC and the exogenous EE-GRSP concentration (Figure 6a) and the DE-GRSP concentration (Figure 6b), respectively.

Figure 5.
Effects of exogenous EE-GRSP and DE-GRSP on soil organic carbon of trifoliate orange seedlings. Data (mean ± SD, n = 4) followed by different letters above the bars indicate significant differences (p < 0.05) between treatments.

Changes in Soil Phosphatase Activity
The control, quarter-, half-, and full-strength EE-GRSP treatments significantly increased the activity of soil acid phosphatase by 20%, 29%, and 10%, respectively, and that

Changes in Soil Phosphatase Activity
The control, quarter-, half-, and full-strength EE-GRSP treatments significantly increased the activity of soil acid phosphatase by 20%, 29%, and 10%, respectively, and that of neutral phosphatase by 21%, 28%, and 86%, respectively (Figure 7a,b). The application of the half-and full-strength EE-GRSP also significantly increased soil alkaline phosphatase activity by 26% and 13%, respectively, with no significant effect caused by the application of the quarter-strength EE-GRSP (Figure 7c). In addition, compared with the control, the quarter-, half-, and full-strength DE-GRSP treatments had no significant effect on the activity of soil neutral and alkaline phosphatase, but the treatments significantly decreased soil acid phosphatase activity by 11%, 13%, and 31%, respectively (Figure 7a-c).

Changes in Soil Phosphatase Activity
The control, quarter-, half-, and full-strength EE-GRSP treatments significantly increased the activity of soil acid phosphatase by 20%, 29%, and 10%, respectively, and that of neutral phosphatase by 21%, 28%, and 86%, respectively (Figure 7a,b). The application of the half-and full-strength EE-GRSP also significantly increased soil alkaline phosphatase activity by 26% and 13%, respectively, with no significant effect caused by the application of the quarter-strength EE-GRSP (Figure 7c). In addition, compared with the control, the quarter-, half-, and full-strength DE-GRSP treatments had no significant effect on the activity of soil neutral and alkaline phosphatase, but the treatments significantly decreased soil acid phosphatase activity by 11%, 13%, and 31%, respectively (Figure 7a-c).

Figure 7.
Effects of exogenous GRSP on soil acid (a), neutral (b), and alkaline (c) phosphatase activity in trifoliate orange seedlings. Data (mean ± SD, n = 4) followed by different letters above the bars indicate significant differences (p < 0.05) between treatments.

Discussion
The present study showed that different concentrations of EE-GRSP dramatically increased shoot and root biomass production, while half-strength EE-GRSP had the most significant effect, which was consistent with the results of Wang et al. [28] and Chi et al. [29] in trifoliate orange seedlings. The EE-GRSP contained high amounts of humic acids, which had functions in improving plant growth [30]. However, different concentrations of DE-GRSP all significantly reduced shoot and root biomass production with increased concentrations, implying that exogenous DE-GRSP negatively affects the growth of trifo-

Discussion
The present study showed that different concentrations of EE-GRSP dramatically increased shoot and root biomass production, while half-strength EE-GRSP had the most significant effect, which was consistent with the results of Wang et al. [28] and Chi et al. [29] in trifoliate orange seedlings. The EE-GRSP contained high amounts of humic acids, which had functions in improving plant growth [30]. However, different concentrations of DE-GRSP all significantly reduced shoot and root biomass production with increased concentrations, implying that exogenous DE-GRSP negatively affects the growth of trifoliate orange seedlings. The different growth responses for exogenous EE-GRSP and DE-GRSP applications could be derived from two facts: (i) the difference in the intrinsic elemental composition between EE-GRSP and DE-GRSP (e.g., higher P, Mo, and O contents in purified EE-GRSP versus purified DE-GRSP; (ii) changes in the synthesis and transport of auxins in plants with higher root indole acetic acid and indole butyric acid in EE-GRSP-treated plants than in DE-GRSP-treated plants [31]. The variation in the intrinsic elemental contents of the two GRSP fractions consisted of different organic substances, such as tyrosine, tryptophan, fulvic acid, humic acid, nitrobenzoxadidole, and calcofluor [32], thus triggers differential plant growth responses. In addition, GRSP is known to contain many impurities as well as proteins of mycorrhizal fungal origin [30,33]. It is possible that EE-GRSP and DE-GRSP always contain different impurities, and these impurities have differential effects on plants; however, the applied EE-GRSP solution (pH 7.0) had a lower pH value than that of DE-GRSP (pH 8.0), and citrus prefers slightly acidic soil.
Soil aggregates are an important constituent of soil structure, and their quantity and stability are the essential criteria for evaluating soil quality [34]. During the formation of soil aggregates, GRSPs, similarly to glue, are involved in binding small soil particles into microaggregates (<0.25 mm in diameter) to large aggregates (> 0.25 mm in diameter), ultimately forming a stable soil aggregate structure [35]. Compared with the control treatment, the quarter-strength EE-GRSP application significantly increased WSA in the sizes of 2-4 mm and 0.5-1.0 mm but reduced WSA in the 1-2 mm and 0.25-0.5 mm sizes. The half-strength EE-GRSP treatment considerably increased WSA in all sizes. The fullstrength EE-GRSP significantly increased WSA in the 2-4 mm, 1-2 mm, and 0.5-1 mm sizes. Wu et al. [23] also reported that exogenous EE-GRSP significantly increased WSA in the sizes of 1-2 mm and 0.5-1 mm in field citrus. Contrastingly, the present study found that treatments with the quarter-, half-, and full-strength DE-GRSP dramatically decreased WSA in all sizes. It was proposed that in GRSP fractions, only EE-GRSP represented the positive effect on WSA formation, whereas DE-GRSP had a negative effect. In general, soil WSA formation is involved in mycorrhizal hyphae, SOC, roots, GRSP, etc. [36]. Wu et al. [37] reported that in the presence of roots and mycorrhizal hyphae, root biomass, and mycorrhizal colonization are the main mechanisms in WSA formation, and total GRSP concentration (the sum of EE-GRSP and DE-GRSP) is of paramount importance in WSA formation.
In this study, we also observed differential effects of exogenous EE-GRSP and DE-GRSP on MWD: the increase caused by EE-GRSP and the decrease caused by DE-GRSP. Correlation analysis indicated that there was a significant curvilinear correlation between MWD and the concentration of exogenous EE-GRSP and DE-GRSP. This suggested that EE-GRSP application had positive effects on WSA stability, whereas DE-GRSP had deleterious effects on WSA stability. Thus, in GRSP fractions, only EE-GRSP contributed to the aggregate stability, as previously reported by Wu et al. [38] under the presence of only mycorrhizal extraradical hyphae conditions. Chi et al. [39] reported that higher soil pH in saline-alkali regions reduced soil aggregate contents and stability. In our study, soil pH value was increased with the increase in exogenous DE-GRSP concentrations and decreased with the increase in exogenous EE-GRSP concentrations. In addition, soil pH value was significantly and negatively correlated with MWD, implying that exogenous GRSP could modulate soil pH value to affect WSA formation and stability, which still needs to be researched in future studies.
SOC content affects the physical and chemical properties of soil, as well as its fertility characteristics, and, thus, it is considered as one of the decisive factors in evaluating soil quality [40]. The present work indicates that the control, quarter-, half-, and full-strength EE-GRSP and DE-GRSP treatments increased the SOC content, which is consistent with our earlier results in a 27-year-old Satsuma mandarin grafted onto a trifoliate orange Agronomy 2021, 11, 1896 9 of 11 after being applied with exogenous EE-GRSP [23]. Additionally, the stimulated effect on SOC was observed under exogenous DE-GRSP application conditions. Studies showed that purified GRSP contributed to 23.26 ± 2.67% of the total SOC [4]. In this study, DE-GRSP had a relatively higher contribution to the SOC than EE-GRSP in the full-strength concentration. This further suggests that DE-GRSP in the full-strength concentration had a stronger contribution to SOC than EE-GRSP. Our previous data also revealed a higher contribution of purified DE-GRSP (14.59 ± 2.21%) versus purified EE-GRSP (8.67 ± 0.95%) to SOC [4].
Soil enzymatic activity is a key indicator of soil biochemical characteristics [41]. Among them, soil phosphatases catalyze the mineralization of soil organophosphorus compounds, and phosphatase activities directly affect the decomposition and transformation of soil organophosphorus [42]. Exogenous EE-GRSP treatments significantly increased soil acidic, neutral, and alkaline phosphatase activity, which was consistent with previous works on Satsuma mandarin trees grown in fields [22]. In contrast, exogenous DE-GRSP mainly reduced soil acid phosphatase activity but did not alter soil neutral and alkaline phosphatase activity. This means that the rhizosphere of EE-GRSP-treated plants possessed stronger acid, neutral and alkaline phosphatase activity, while the rhizosphere of DE-GRSP-applied plants had lower acid phosphatase activity; this change may correlate with differential plant growth triggered by GRSP applications.

Conclusions
In conclusion, the application of exogenous EE-GRSP considerably increased plant biomass, stimulated the formation and stability of soil WSAs, reduced soil pH value, and elevated SOC content and soil phosphatase activity. This indicates that exogenous EE-GRSP can be considered as a biostimulator for the improvement of both plant growth and soil properties. Furthermore, we also observed that exogenous DE-GRSP exhibited adverse effects on plant biomass, WSA formation and stability, soil pH, and soil acid phosphatase activity. Such results guide the management of soil GRSP in citrus orchards for reducing DE-GRSP and increasing EE-GRSP.