Next Article in Journal
Biocontrol Efficacy of Bacillus thuringiensis Strain 00-50-5 Against the Root-Knot Nematode Meloidogyne enterolobii in Pepper
Previous Article in Journal
A Comparative Analysis of Ethnic Networks and Internationalization of Latin American Agri-SMEs: The Case of Argentina, Brazil, and Chile
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agriculture
Volume 14
Issue 11
10.3390/agriculture14111919
agriculture-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Effect of P Reduction on phoD-Harboring Bacteria Community in Solar Greenhouse Soil

by
Ting Bian
1,2,3,4,†,
Zhen Wang
1,2,3,4,†,
Shuang Wang
1,2,3,4,
Xuan Shan
1,2,3,4,
Tianqi Wang
1,2,3,4,
Hongdan Fu
1,2,3,4,* and
Zhouping Sun
1,2,3,4,*
1
College of Horticulture, Shenyang Agricultural University, Shenyang 110866, China
2
Key Laboratory of Protected Horticulture of the Education Ministry and Liaoning Province, Shenyang 110866, China
3
National & Local Joint Engineering Research Center of Northern Horticultural Facilities Design & Application Technology, Shenyang 110866, China
4
Collaborative Innovation Center of Protected Vegetable Suround Bohai Gulf Region, Shenyang 110866, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agriculture 2024, 14(11), 1919; https://doi.org/10.3390/agriculture14111919
Submission received: 14 September 2024 / Revised: 22 October 2024 / Accepted: 25 October 2024 / Published: 29 October 2024
(This article belongs to the Special Issue Enzymatic Activity and Functional Diversity of Soil Microbial Communities in Agricultural Soils)
Browse Figures
Versions Notes

Abstract

:
Phosphorus (P) enrichment frequently occurs in the soil used in greenhouse vegetable production (GVP). Minimizing the application of P fertilizer represents a crucial approach to mitigating the accumulation of P in the soil and enhancing its utilization efficiency. However, the changes in bacterial communities and the turnover mechanism of soil P fractions related to soil P cycling after P fertilizer reduction are still unclear. To unravel these complexities, we devised three experimental treatments: conventional nitrogen (N), P, and potassium (K) fertilizer (N1P1K1); conventional N and K fertilizer without P (N1P0K1); and no fertilizer (N0P0K0). These experiments were conducted to elucidate the effects of P reduction on cucumber plant growth, soil P fractions, and the phoD-harboring bacterial community in the P-rich greenhouse soil. The results showed that there were no significant differences between the N1P1K1 and N1P0K1 treatments in terms of plant growth, yield, and P uptake, and the values for the N0P0K0 treatment were significantly lower than those for the N1P1K1 treatment. In a state of P depletion (N0P0K0, N1P0K1), the main P sources were Resin-Pi, NaHCO3-Pi, NaHCO3-Po, and NaOH-Pi. The contents of NaOH-Po and CHCl-Po in the N1P0K1 treatment increased significantly. Without P fertilizer, alkaline phosphatase (ALP) activity, phoD gene abundance, and bacterial community diversity were significantly increased. The abundance of Ensifer in the N0P0K0 and N1P0K1 treatments was 8 and 10.58 times that in the N1P1K1 treatment, respectively. Additionally, total phosphorus (TP) and available nitrogen (AN) were key factors affecting changes in the phoD bacterial community, while Shinella, Ensifer and Bradyrhizobium were the main factors driving the change in soil P fractions, and NaHCO3-Pi and NaOH-Pi were key factors affecting crop yield. Therefore, reducing the application of P fertilizer will increases the diversity of phoD-gene-harboring bacterial communities and promote organic P mineralization, thus maintaining the optimal crop yield.

1. Introduction

P is one of the essential nutrients for plant growth and development, and it is the main limiting factor for stable and increased yields [1]. Over the last two decades, GVP in China has undergone significant development, playing a pivotal role in fulfilling the demand for fresh vegetables, particularly during the winter season. Under the influence of economic interests and farmers’ own irrigation and fertilization practices, there is a widespread problem of excessive resource input and excessive fertilizer use (especially P fertilizer) in greenhouse vegetable cultivation. The average single-season chemical P fertilizer dosage for greenhouse vegetables in China is 1308 kg·hm−2, which is 13.0 times the amount of P required by vegetables [2]. The excessive application of P fertilizers causes a nutrient imbalance in plants, resulting in premature crop maturity, smaller grains, lower quality, and lower yields [3]. The prolonged application of excessive amounts of P fertilizer not only constitutes a waste of P resources and elevates production costs but also contributes to a decline in the efficiency of P fertilizer utilization, culminating in a significant accumulation of P [4]. P exhibits a robust soil fixation capability, and the residual effects of a single large application of P fertilizer can persist for a minimum of 10 years [5]. However, the capacity for P adsorption in the soil is constrained, and the conventional application of excessive P fertilizer surpasses the soil’s adsorption threshold, leading to a substantial loss of P nutrients. Consequently, in agricultural systems, it is imperative to utilize limited but optimal P resources in a judicious manner.
Several researchers in the United States and Australia have concluded that reduced P fertilizer application in open-field soils has a significant effect on improving P fertilizer utilization, mitigating the phenomenon of P accumulation and reducing the potential threat of environmental pollution [6,7]. A study found that reducing the application of chemical P fertilizer by 20% on the basis of conventional P application for five consecutive years did not reduce the available phosphorus (AP) content and yield of maize soil [8]. However, crop yield decreased from 6.1 t·ha−1 to 5.1 t·ha−1 with a reduction in both TP and AP content when nitro nitrogen (N), P and potassium (K) fertilizers were not applied to wheat–maize rotation soil from 2001 to 2016 [9]. In addition to TP and AP, which characterize the overall level of soil P and the level of soil P utilized by plants, soil P fractions can more clearly reveal the P supply capacity of soil to plants. Numerous scholars have embraced the Hedley P fractional extraction method revised by Tissen to characterize soil P fractions. In this method, Resin-Pi, NaHCO3-Pi, and NaHCO3-Po were classified into labile P, which was highly effective and could be directly utilized by plant microorganisms. NaOH-Pi, NaOH-Po, and Dil. HCl-Pi are classified as moderately labile P, which cannot be directly utilized but can be used as a potential phosphorus source for plants and microorganisms. Concentrated HCl-Pi, concentrated HCl-Po, and residual-P provide non-labile P [10,11]. Nishigaki found that labile inorganic phosphorus (Pi) was the main source of the P pool for P uptake by rice crops in treatments both without chemical P fertilizer (+NK) and without N, P, and K fertilizer [12]. Some scholars believe that NaHCO3-Po and NaOH-Po can be regarded as the main sources of AP for plants under the condition of reducing P fertilizer use [13]. Numerous studies have demonstrated that a suitable reduction in the application of P fertilizer in GVP not only fails to hinder crop growth and yield but also enhances yield and fruit quality [14]. When P fertilizer was reduced in greenhouse tomato soil for four consecutive crops, the AP content showed a decreasing trend, with an average decrease of 3.4 mg·kg−1 per crop; compared with that of a conventional P fertilizer treatment, the TP content of GVP soil decreased by 19.8% [15]. Nonetheless, there is scant understanding of the importance of these varied P fractions in the P supply for crops cultivated in greenhouse soils with diminished P fertilization.
Microorganisms are instrumental in regulating the conversion process of soil organic P (Po). Notably, the phoD gene, which is the most pivotal ALP gene present in soil, exerts a significant influence on the bioactivation of soil Po [16]. When the content of soil AP is low, soil microorganisms secrete ALP, which can dephosphorylate organic matter (SOM), remove the phosphate group on the substrate molecule under the action of hydrolyzed phosphate ester, and generate orthophosphate for the absorption and utilization of plants [17].
There are a large number of P-solubilizing microorganisms in nature, and the number of P-solubilizing bacteria accounts for 1–50% of the total number of soil microorganisms. The known bacteria with strong Po activation ability are mainly Mesorhizobium, Bradyrhizobium, Sinorhizobium, and Pseudomonas. Many studies have shown that different fertilization strategies change the community composition and diversity of phoD-gene-harboring bacteria [18,19]. For example, in field soil, excessive application of P fertilizer was whown to inhibit phosphatase activity by reducing the α-diversity of the phoD-gene-harboring community in maize soil [20]. By contrast, diminishing the utilization of P fertilizer can elevate the absolute abundance of the phoD gene and the relative abundance of Bradyrhizobium and Methylobacterium within the associated bacterial community, which, in turn, promoted phosphatase hydrolysis [21]. In wheat–maize rotation soil, compared with the continuous application of P fertilizer, it was found that phoD gene abundance significantly increased without P fertilizer treatment, soil ALP activity was induced, and soil Po mineralization was accelerated [22]. Research indicates that ALP activity has a direct effect on NaOH-Po, and NaOH-Po directly affects the change in NaOH-Pi content [23]. Yu et al. also found that after long-term rice cultivation, the ALP secreted by microorganisms mainly mineralized labile Po and moderately labile Po [24]. This indicated that the phoD-gene-harboring bacterial community was intimately associated with alterations in the phosphatase content and P fraction. Previous studies have focused on phoD-gene-harboring bacterial communities in dryland or paddy soils. The cumulative amount of P in GVP soil is higher than that in other planting systems. However, the combined effect of the community of bacteria with the phoD gene and ALP activity on P activation in GVP soils under P fertilizer reduction is still unclear.
Hence, the present study focuses on the problem of the excessive application of P fertilizer in the GVP. A pot experiment was used to study the effects of reduced application of P fertilizer in four consecutive plots and reductions in P fertilizer in combination with other fertilizers on the growth, yield, and P content absorbed by cucumber plants in the GVP. The effect mechanisms of the effects of two kinds of continuous P reduction on P fractions, phoD-gene-harboring bacterial community structure, and ALP activity in the GVP soil, as well as the effect on cucumber yield, were clarified in order to provide theoretical support for a scientific P reduction strategy in the production of greenhouse cucumbers and improve the utilization of soil Po mineralization.

2. Materials and Methods

2.1. Experimental Design and Soil Collection

In this study, the test soil was taken from a greenhouse in Yuantai Town, Wafangdian City, Liaoning Province, in which cucumber had been grown for 10 years. The tested soil was brown soil, and its basic properties and P fractions are shown in Table 1. The P reduction experiment began at the end of August 2019. First, 6 kg of test soil were positioned within a plastic pot, featuring an upper internal diameter of 41 cm, a lower internal diameter of 30 cm, and a height of 28 cm. Subsequently, cucumber seedlings aged 26 days were transplanted into this pot.
Solar greenhouse cucumbers have two growing seasons (two rounds a year), each lasting 110 days. In this experimental study, a systematic approach was adopted to design three distinct fertilization gradients, utilizing urea, potassium dihydrogen phosphate, and potassium sulfate as the primary sources of N, P, and K fertilizers, respectively. Conventional application of N, P and K fertilizer (N1P1K1) was used as control, and conventional application of N and K fertilizer, no P fertilizer (N1P0K1) and no N, P and K fertilizer (N0P0K0) were used as treatments. The control experiment entailed the conventional application of N, P, and K fertilizers (N1P1K1), whereas the treatment experiments comprised two scenarios: one involved the conventional application of N and K fertilizers without P fertilizer (N1P0K1), and the other entailed the complete absence of N, P, and K fertilizers (N0P0K0). Each treatment was configured with three replicate samples, randomly distributed in the solar greenhouse. The specific application amount was as follows: P fertilizer (KH2PO4) dosage was 83 kg P2O5 ha−1 round−1, N1P1K1 and N1P0K1 treatment were applied with the same amount of N fertilizer (334 kg N ha−1 round−1) and K fertilizer (234 kg K2O ha−1 round−1), respectively, all fertilizer amounts are meticulously calculated in accordance with the precise production levels of local farmers. One third of the total fertilizer mix was applied in the form of water-soluble fertilizer at 30, 50 and 70 days of cucumber colonization. In addition to the amount of fertilizer applied, other cultivation and management measures remain consistent. We conducted four rounds cucumber weight loss experiments from the end of August to December 2019 (first round), the end of February to June 2020 (second round), the end of August to December 2020 (third round) and the end of February to June 2021 (fourth round).
Soil samples (0–10 cm) from three treatments were collected with a soil extractor at the time of pulling the fourth round of cucumber planting (June 2021). The soil sample was subjected to a rigorous 2 mm sieving process and subsequently divided into three distinct sections. One section was meticulously air-dried in a cool and controlled environment and then utilized for a comprehensive analysis of soil physical and chemical properties, as well as the determination of the P fractions. Another section was stored under strictly regulated conditions at 4 °C, with the intention of conducting soil enzyme activity tests within 1 week. Lastly, the remaining part was preserved at a temperature of −80 °C, ensuring optimal conditions for the determination of phoD harboring bacterial community composition and abundance.

2.2. Measurement of Morphological Traits, Yield and Nutrient Content of Cucumber Plants

On the 30th, 60th, and 90th days subsequent to planting, three cucumber plants were systematically and randomly sampled from each treatment group in order to accurately assess their plant height, which is defined as the vertical distance extending from the cotyledon to the cucumber’s growing point. Three cucumber plants were randomly selected for each treatment, and continuous yield measurements were made with an electronic balance as the cucumber fruits matured. Records were kept, and the average yield of single cucumber plants was counted after pulling the seedlings. Yield was measured by the total fresh weight of the fruit. The plants were sampled during the seedling pulling period. The roots, stems and leaves of the plants were individually excised and placed in an oven, and the dry weight of each component was measured after drying at 80 °C. The total P content in the roots, stems, leaves, and fruits of the plants was subjected to digestion and extraction using the H2SO4-H2O2 digestion method. Subsequently, the concentration of P in the extracted solution was accurately determined through the molybdenum-blue colorimetric method, as per established protocols [25].

2.3. Soil Physical and Chemical Properties Analyses

Soil pH was determined utilizing a soil:water suspension (1:2.5 w/v). The determination of SOM was conducted through potassium dichromate method. Soil total nitrogen (TN), TP, and total potassium (TK) were quantified using Kjeldahl nitrogen determination, molybdenum blue oil colorimetry, and flame spectrophotometry, respectively. Soil total carbon (TC) was determined with an elemental analyzer (EA3000, EuroVector, Pavia, Italy). Soil AN, AP, and available potassium (AK) were measured by employing the alkaline hydrolysable reduction diffusion method, molybdenum blue colorimetry, and flame spectrophotometry, respectively. The contents of total soil elements, namely calcium (Ca), iron (Fe), and aluminum (Al), were determined through the use of an Inductively Coupled Plasma Emission Spectrometer (ICP Optima 3000 Perkin Elmer, Waltham, MA, USA) [26].

2.4. Extraction of Soil Phosphatase Activity and P Fractions

Using the Hedley P classification method modified by Tissen, we weighed 0.5 g air-dried soil sample (100 mesh) and subsequently placed it into a 50 mL centrifuge tube. Subsequently, we added deionized water and an anion resin membrane; 0.5 M NaHCO3, 0.1 M NaOH and 1 M HCl solution were used for continuous extraction of P fractions (weak to strong) in the soil. Finally, residual-P was extracted by high-temperature digestion with H2SO4 and H2O2. Nine P fractions were obtained in this method: Resin-Pi, NaHCO3-Pi, NaHCO3-Po, NaOH-Pi, NaOH-Po, Dil. HCl-Pi, Conc. HCl-Pi, Conc. HCl-Po and Residual-P. The content of Pi in the solution was determined by molybdenum-blue colorimetric method; the total P (Pt) was determined by colorimetry after adding ammonium persulfate and digested with H2SO4, and the difference between the two was the content of Po [27,28].
Soil acid phosphomonoesterase (ACP) and ALP, phosphodiesterase (PDE) activity was determined by the Tabatabai method [29]. First, a 1 g soil sample was weighed. We then used p-nitrobenzene sodium phosphate as the substrate (ACP) buffer solution (pH 6.5, ALP buffer solution pH 11) and incubated the sample at 37 °C for 1 h. We next weighed out a 1 g soil sample and utilized p-nitrobenzene sodium phosphate as the substrate (ACP) in a buffer solution adjusted to pH 6.5, and an ALP buffer solution adjusted to pH 11. We incubated the mixture at 37 °C for a duration of 1 h. The ACP and ALP activities were determined using UV spectrophotometer (UV-2600i, Shimadzu, Kyoto, Japan) at 410 nm. Next we weighed a further 1 g soil sample and used sodium bis-p-nitrophenyl phosphate as a substrate in a pH 8.0 buffer solution. The mixture was then incubated at 37 °C for 1 h. Subsequently, the PDE activity was also measured utilizing a UV spectrophotometer at 410 nm.

2.5. DNA Extraction, PCR Amplification and Illumina Miseq Sequencing of Soil phoD Community

Soil DNA was extracted from 0.5 g of fresh soil samples utilizing the FastDNA SPIN Kit (MP Biomedicals, Irvine, CA, USA). The phoD gene fragment was amplified utilizing the ABI GeneAmp 9700 PCR system (ABI, Waltham, MA, USA). The primers employed were ALPS-F730 (5′-CAGTGGGACGACCACGAGGT-3′) and ALPS-R1101 (5′-GAGGCCGATcGGCATGTCG-3′), resulting in an amplified fragment size of 371 bp. Each PCR reaction mixture consisted of 20 μL, comprising 4 μL of 5xFastPfu Buffer, 2 μL of dNTPs, 0.8 μL of each primer (ALPS-F730 and ALPS-R1101), 0.4 μL of FastPfu Polymerase, 0.25 μL of BSA, 10 ng of Template DNA, and ddH2O to make up the total volume to 20 μL. The PCR amplification protocol entailed an initial denaturation step at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 45 s. This was concluded with a final extension step at 72 °C for 10 min and subsequent cooling to 10 °C [30,31].
The PCR products were retrieved using 2% agarose gels, subsequently purified with the AxyPrepDNA gel extraction kit. Following purification, the products were eluted with Tris-HCl and subjected to detection via 2% agarose electrophoresis. For sequencing purposes, the Miseq PE300 platform from Meiji Biomedical Technology Co., Ltd. (Shanghai, China) was employed.

2.6. Statistical Analyses

In this research endeavor, the statistical software SPSS 26.0 was employed to meticulously analyze the physiological indices of cucumber, soil physicochemical attributes, P fractions, and the phoD-harboring bacterial community. One-Way Analysis of Variance (ANOVA) was conducted, utilizing the Least Significant Difference (LSD) method. Subsequently, utilizing the R language (3.3.1) in conjunction with the vegan software package, a Principal Component Analysis (PCA) was executed on the phoD-harboring bacterial community under P reduction conditions. To visualize the data, OriginPro 2022 software was utilized to generate heat maps depicting cucumber plant morphology, nutrient composition, P fractions, phosphatase activity, and the phoD-harboring bacterial community at the generic taxonomic level. Furthermore, WPS Excel software 2022 was employed to create a graphical representation of cucumber plant P absorption and soil physicochemical properties. Additionally, Canoco 5.0 software was leveraged to conduct Redundancy Analysis (RDA), aiming to explore the intricate correlations between soil phoD-harboring bacterial community, physicochemical factors, and P speciation. Lastly, Variance Partitioning Analysis (VPA) was performed to investigate the relationship between P fractions and the physiological indices of cucumber.
In this rigorous study, the Fastp software (version 0.20.0) was employed for the purpose of ensuring the quality control of the original sequencing sequence. Subsequently, the FLASH software (v1.2.7) was utilized to splice the sequences, wherein bases with a mass value less than 20 at the terminal end of the read segment were subjected to screening, and sequences failing to meet the prescribed quality standards were excluded (with a maximum allowable mismatch ratio of 0.2). The UPARSE process was then implemented to group the sequences into centrally operated taxonomic units (OTUs). Based on a similarity search of the phoD sequence data within the GenBank database, sequences exhibiting 97% similarity were categorized as belonging to the same OTU, resulting in the identification of 729 distinct OTUs. From each OTU, a representative sequence was selected, and each of these representative sequences was assigned a classification value utilizing the RDP classifier available on the Qiime platform, with a confidence threshold set at 0.7. Statistical analysis was conducted using the OTU samples from Usearch (version 7.0), and both α- and β-diversity were analyzed employing the Mothur software (v.1.36.1). All sequencing data has been deposited in the NCBI Sequence Read Archive (SRA) database, under the accession number PRJNA1169104. To construct the network, OTUs with a relative abundance exceeding 0.01% were selected, and the correlation coefficient was computed utilizing the SparCC correlation matrix. Following the screening of relevant data, the network was constructed with a correlation threshold of 0.6 and a statistical significance level of p < 0.05. The R language (version 3.3.1) software was utilized to visualize the co-occurrence network, enabling the further derivation of the network’s topological properties.

3. Results

3.1. Effects of Reduced P on Morphological Characteristics, Yield and Nutrients of Cucumber Plants in Solar Greenhouse

The N1P1K1 treatment served as the control (CK). Figure 1 reveals that there was no statistically significantly disparity in cucumber plant height, yield, or phosphorus uptake between the N1P0K1 and CK treatments. However, the cucumber biomass in N1P0K1 was notably elevated compared to CK. In contrast, N0P0K0 exhibited a marked decrease in cucumber plant height, yield, biomass, and P uptake when compared to CK. The findings underscore that N1P0K1 significantly surpassed N0P0K0 in terms of cucumber plant height, yield, biomass, and phosphorus uptake.
Compared with CK, the P uptake and distribution ratio in the stems of the N1P0K1 treatment were significantly decreased. P uptake in stems, leaves and fruits of the N0P0K0 treatment showed a significant decrease, P allocation ratio in leaves was significantly increased, and P allocation ratio in fruits was significantly decreased. Compared with N1P0K1, it was found that the P uptake in leaves and fruits of N0P0K0 was significantly reduced, while the P allocation proportion in leaves was significantly increased and the P allocation proportion in fruits was significantly decreased (Table S1).

3.2. Effects of P Fertilizer Reduction on Soil Chemical Properties of Cucumber in Solar Greenhouse

As shown in Table 2, compared with CK, N1P0K1 and N0P0K0 significantly reduced the contents of TN, TP, AP and AK. Except for AK, there were no significant differences in other indicators between the two treatments. The AN content of N1P0K1 was significantly higher than CK and N0P0K0. There was no significant difference between N1P0K1, N0P0K0 and CK in pH, TK, SOM and M3-Al. The contents of M3-Ca and M3-Fe in the three treatments were all CK< N1P0K1< N0P0K0.

3.3. Effects of P Fertilizer Reduction on Soil P Fractions and Enzyme Activities of Cucumber in Solar Greenhouse

As shown in Figure 2, reducing the application of P fertilizer affected the soil P fractions. Compared with CK, N1P0K1 and N0P0K0 showed significantly reduced soil labile P (Resin-Pi, NaHCO3-Pi, NaHCO3-Po), moderately labile P (NaOH-Pi, Dil. HCl-Pi) and non-labile P (Conc. HCl-Pi, Conc. HCl-Po) contents. The contents of NaHCO3-Pi, NaOH-Pi, and Conc. HCl-Po in N1P0K1 were significantly higher than N0P0K0. There was no significant difference in NaOH-Po between N1P1K1 and N1P0K1. Compared with N1P1K1, N1P0K1 and N0P0K0 treatments significantly increased NaOH-Po/TPo, NaOH-Pi/TPi, Dil. HCl-Pi/TPi and Conc. HCl-Pi/TPi and significantly decreased Resin-Pi/TPi (Tables S2–S4).
Compared with N1P1K1, ALP activity was increased by 80.14% and 66.55%, and PDE activity was increased by 69.12% and 38.66% in N1P0K1 and N0P0K0, respectively (Figure 3).

3.4. Effects of P Fertilizer Reduction on Soil phoD Gene Abundance and Community Diversity of Cucumber in Solar Greenhouse

Reducing P fertilizer affected soil phoD gene abundance; the phoD gene abundance of N1P0K1 and N0P0K0 were significantly higher than CK by 46.95% and 25.00% (Figure 4a). The sequences obtained after high-throughput sequencing and quality control with 97% similarity were aligned and annotated with the NT database. Shannon index was used to represent the phoD bacterial community diversity. The Shannon indexes of the N1P1K1, N1P0K1, and N0P0K0 treatments were 3.46, 4.62, and 4.11, respectively. The Shannon indexes of the N0P0K0 and N1P0K1 treatments were significantly higher than that of the N1P1K1 treatment (Figure 4b). According to the PCA analysis conducted, it was observed that various P fertilizer reduction treatments exerted a notable impact on the phoD-harboring bacterial communities. The first component analysis yielded an explanatory degree of 23.53%, revealing a statistically significant difference between the N0P0K0 and the N1P0K1 treatments as well as N1P1K1 treatments. Furthermore, the second component analysis demonstrated a 14.56% explanatory degree, with a significant distinction observed between the N1P0K1 treatment and the N0P0K0 as well as N1P1K1 treatments (Figure 4c).

3.5. Effects of P Fertilizer Reduction on Soil phoD-Harboring Bacterial Community of Cucumber in Solar Greenhouse

The proteobacteria phylum emerged as the dominant phoD-harboring phylum, accounting for 15.38% to 22.28%. Within this dominant phylum, no notable variation in abundance was observed between the N1P0K1 and N1P1K1 treatments. However, the abundance of proteobacteria in the N0P0K0 treatment was markedly lower compared to the N1P0K1 and N1P1K1 treatments (Table S5). Figure 5 presents an analysis of the species composition and sample cluster tree of the top 12 total abundance of phoD-harboring bacterial communities at the genus level within the soil of the greenhouse cucumber under reducing P fertilizer treatments. The findings indicate a greater similarity in the dominant genera composition of the soil under N1P1K1 and N1P0K1 treatments. In the context of the phoD-harboring bacterial community, the prevalent genera were Bradyrhizobium (10.63–14.91%), Shinella (0.01–3.08%), and Ensifer (0.12–1.27%) (Figure 6). A comparative assessment with the N1P1K1 treatment revealed that both the N1P0K1 and N0P0K0 treatments significantly decreased the abundance of Bradyrhizobium and Shinella while significantly elevating the abundance of Ensifer. An analysis of the network’s topological properties showed that network node connectivity in the N1P0K1 and N0P0K0 treatments was higher than N1P1K1 treatment (Figure 7).

3.6. Effects of Reducing P Fertilizer on Soil Properties and the Relationship between P Fractions and the phoD-Harboring Bacterial Community of Cucumbers in Solar Greenhouses

RDA results showed that in the N1P1K1 and N1P0K1 treatments, the two canonical axes explaining the soil physicochemical properties of the phoD-harboring bacterial community were 92.81% and that soil TP was an important factor affecting the phoD-harboring bacterial community (Figure 8a). In the N1P1K1 and N0P0K0 treatments, the two canonical axes explaining of soil physicochemical properties in the phoD-harboring bacterial community were 90.70% and soil AN was an important factor affecting the phoD-harboring bacterial community (Figure 8b). The Ensifer was a factor that significantly influenced the changes of soil P morphology in the N1P1K1 and N1P0K1 treatments (Figure 9a). Shinella, Sinorhizobium, and Bradyrhizobium were factors that significantly affected soil P morphology changes in the N1P1K1 and N0P0K0 treatments (Figure 9b). In the Pearson correlation analysis carried out to evaluate the relationship between dominant genera and P fractions, it was found that Shinella showed a statistically significant positive correlation with all P fractions except Residual-P. In contrast, Ensifer exhibited a significant negative correlation with NaOH-Po. Moreover, Bradyrhizobium was discovered to have a statistically significant positive correlation with Conc. HCl-Po (Table S6).
The impact of soil P fractions and physicochemical properties on the variation of cucumber physiological indexes was analyzed by VPA. It was found that all the above indexes explained 50.7% and 45.2% of the variation of cucumber physiological indicators, respectively, and the soil P fractions had the highest explaining degree (Figure 10a). The random forest model was also used to identify key P fractions affecting cucumber yield. NaHCO3-Pi (7.97%) had the highest MPI, followed by NaOH-Pi (7.82%), Conc. HCl-Po (6.28%) and Resin-Pi (4.25%) (Figure 10b).

4. Discussion

4.1. Changes in the Soil phoD-Harboring Bacterial Community and Its Relationship with Soil Physical and Chemical Properties

Soil microorganisms can be used to indicate the availability of soil P effectiveness or plant nutrient limitation, and the phoD-harboring bacterial taxon encoding ALP plays an important role in the release and transformation of soil Po. Studies have shown that ALP activity is sensitive to the availability of P, because its synthesis depends on the phosphate content, reflecting the availability of phosphate to microorganisms [32]. Increased ALP activity indicates that microorganisms require a large amount of energy to mineralize Po [33]. In this study, the N1P0K1 and N0P0K0 treatments significantly reduced soil AP and TP content compared with the N1P1K1 treatments, significantly increased the soil phoD gene abundance, and significantly increased the ALP activity (Figure 3 and Figure 4). The N1P0K1 and N0P0K0 treatments alleviated microbial P restriction, and the increase in microbial quantity promoted the production of ALP, while the N1P1K1 treatment had higher Pi effectiveness, which inhibited the production of ALP. The reduction in P fertilization had a discernible impact on the diversity of the soil phoD-harboring bacterial community diversity. Specifically, the Shannon of phoD in the N1P0K1 treatment was significantly higher than that in the N1P1K1 treatment (Figure 4). This may have been due to the fact that test soil was P-rich, and continuous application of P fertilizer will inhibit plant growth, thus inhibiting the process of P dissolution. However, reduced application of P fertilizer can promote plant growth, allowing more organic substrates to be secreted and stimulating the growth of more low-abundance microbial species [34]. Interestingly, both the phoD gene abundance and the α-diversity of the phoD-harboring bacterial community were higher in the N1P0K1 treatment than in the N0P0K0 treatment. This observation suggests that the application of N and K fertilizers affected the phoD-harboring bacterial community’s composition. Furthermore, the application of N fertilizer has the potential to indirectly enhance the diversity of this specific bacterial community via the process of soil acidification. Wang et al. showed that soil TK can also significantly affect phoD-harboring bacterial communities, but the mechanism by which TK affects phoD-harboring bacterial communities is unclear [35].
The phoD-harboring bacterial community showed that its dominant species were mainly distributed between Proteobacteria and Actinobacteria (Table S2), which has been similarly reported in previous studies. Studies have conclusively demonstrated that Pseudomona and Massilia represent the most significant taxonomic groups that facilitate the mineralization of Po in wheat soil with low P content [36]. Mitsuaria and Kribbella are the main taxa that promote Po mineralization in soils with sufficient-P, indicating that different P supply levels regulate the changes in key taxa in soils [37]. Bradyrhizobium was the dominant species in the phoD-harboring bacterial community in this study (Figure 5), and it plays an important role in the plant–microorganism interactions. Specifically, Bradyrhizobium is a nitrogen-fixing symbiotic organism requiring phosphate transport systems, and it can produce phosphatases or organic acids to improve soil P availability [38]. Bradyrhizobium is a eutrophic bacterium with high nutrient requirements. It participates in plant–microbial interactions, which explains why the abundance of Bradyrhizobium decreases with reduced P fertilizer application. In this study, it was found that reduced P fertilizer application significantly increased the abundance of soil Ensifer, Sinorhizobium and Nitratireductor (Figure 5). Enisifer and Sinorhizobium are most commonly found to be enriched in the rhizosphere of maize; they are capable of forming symbiotic relationships with legumes and are also prevalent in non-legumes such as cucumbers or eggplants [39]. They all belong to the group of nitrogen-fixing symbiotic bacteria and require a phosphate transport system; thus, they can activate Pi and Po by secreting organic acids or phosphatases while fixing nitrogen. Thus, compared with continuous application of P fertilizer, no P fertilizer treatment can promote the growth of nitrogen-fixing bacteria (such as Ensifer, Sinorhizobium and Nitratireductor), thus improving the availability of soil P. Our analysis of the network topological properties showed that the number of nodes and connections of the phoD-harboring bacterial community network structure increased significantly after four consecutive crops without P fertilization in P-rich soil (Figure 6), indicating that the N1P0K1 and N0P0K0 treatments promoted the interspecific competition of the phoD bacterial community and tended to be complicated. The findings from RDA conducted on soil nutrients under various treatments, which served serving as environmental factors, and distinct microbial communities indicated that the phoD-harboring bacterial community underwent notable variations in response to TP in the N1P1K1 and N1P0K1 treatments. Furthermore, the phoD-harboring bacterial community in the N1P1K1 and N0P0K0 treatments exhibited significant changes in response to AN (Figure 7). In soil systems, N and P dynamics are interrelated, and N has a direct or indirect effect on P conversion by influencing microbial or phosphatase activity [40]. Although the AN content of the N0P0K0 treatment (38.5 mg·kg−1) was slightly lower than that of N1P1K1 the treatment (41.61 mg·kg−1), there were no significant differences between the two treatments, and the phoD gene abundance in the N0P0K0 treatment was significantly higher than that in the N1P1K1 treatment, indicating that the original accumulation of AN in the soil may have stimulated P-soluble microorganisms and, thus, promoted the input of resources for the microbial community under the condition of four consecutive crops without N fertilizer to enhanced the P cycle’s potential. The transformation of TP in soil involves dissolution, adsorption, fixation, and mineralization, etc. In this study, after planting four cucumbers crops wer planted, the TP in the N1P0K1 treatment decreased significantly by 8.79%, which was mainly the result of P absorption and consumption by cucumber and microorganisms during the growing period of the crops. TP was significantly positively and negatively correlated with the abundance of Shinella and Ensifer bacteria (Table S5). These results indicated that the change in TP is closely related to the regulation of the phoD-gene-harboring microbial community. These results are consistent with those of Zheng, who suggested that TP and AP have an effect on organophosphate-mineralizing microbial communities, among which Stenotrophomonas, Variibacter and Bradyrhizobium are significantly associated with AP [41]. Relevant studies have shown that N is a crucial component for microbial proliferation, and any alterations in its concentration have the potential to impact the establishment and composition of bacterial communities.

4.2. The Soil P Fraction and Its Relationship with the phoD-Harboring Community

Soil AP content serves as a crucial indicator of the soil’s P supply capacity, and it can reflect the ability of plants to directly absorb P in the current season. The TP content denotes the aggregate amount of P stored within the soil, mirroring the magnitude of the soil P reservoir [42]. In this study, it was ascertained that the primary source of P for plants was the soil legacy P in the N1P0K1 and N0P0K0 treatments. In contrast, the continuous application of P fertilizer increased the soil TP and AP content. After the cultivation of four consecutive crops, our analysis revealed that in comparison with the original soil composition, the contents of soil TP and AP exhibited a decline under the N0P0K0 and N1P0K1 treatment regimes. Conversely, the N1P1K1 treatment demonstrated a notable enhancement in the AP content by 49.29% and in the TP content by 15.75%, respectively, as depicted in Table 2. This was in accordance with prior research findings. In the loamy, sandy soil of India, the annual decline in soil AP content averaged 0.89–4.98 mg·kg−1 per year without the application of P fertilizer for three years [43]. In the black soil of China, the content of soil TP under P application increased by 53.9–65.7% compared with that under no P fertilizer application, and the content of AP in cotton soil under P application was 1.8 times higher than that without P fertilizer application [44].
Research has indicated that soil Pi serves as the primary source for P uptake by plants, and alterations in its content and morphology can serve as indicators of the soil’s P availability [45]. In this study, soil P fractions were determined using the Hedley P extraction method revised by Tissen. After four crops in pot trials, it was found that the contents of labile Pi (Resin-Pi and NaHCO3-Pi) and moderately labile Pi (NaOH-Pi and Dil. HCl-Pi) were significantly reduced in the N1P0K1 and N0P0K0 treatments, indicating that these four P fractions were the main sources of the P absorbed and utilized by plants (Figure 2). The reason for this was that Resin-Pi and NaHCO3-Pi represent the most effective soluble Pi for plants, and they can be directly absorbed and utilized by crops [46]. NaOH-Pi is Pi that has been adsorbed on amorphous aluminum–iron oxides; it is considered as moderately labile P and can also be used by plants after desorption. Dil. HCl-Pi is a calcium-bound compound, and it is mainly related to primary apatite minerals. Both Dil. HCl-Pi and NaOH-Pi are moderately labile Pi, accounting for a large proportion of TP, and they have high P activity. Under the condition of a long-term lack of P application, they can be converted into other forms as a source of P for crop uptake and use under long-term no P application [47]. Continued application of P in P-rich soil will cause a large amount of soil Pi to accumulate, mainly through the accumulation of Resin-P. This finding is congruent with prior research that found that a large amount of labile P is released when chemical P fertilizer is applied to the soil [48].
Soil Po has strong mobility and weak fixation. It can only be used by plants after decomposition into Pi through mineralization. NaHCO3-Po is mainly a soluble Po compound and some microbial P, and it is easily mineralized and used by plants [49]. In the current research, the treatment without P fertilizer markedly decreased the NaHCO3-Po content compared with that in the four continuous cropping rounds with the P fertilizer application treatment. This indicated that the soil NaHCO3-Po was mineralized to NaHCO3-Pi for plant uptake and utilization after the application of fertilizer with no P. This study found that the main form in which soil Po occurred was NaOH-Po (Figure 2 and Table S2), and the content of NaOH-Po in the N1P0K1 treatment had no significant difference from the N1P1K1 treatment, but it was significantly higher than the N0P0K0 treatment. This indicated that the application of N fertilizer could promote an increase in the NaOH-Po content and then promote the mineralization of NaOH-Po into NaOH-Pi, which explains why the NaOH-Pi content in the N1P0K1 treatment was significantly higher than N0P0K0 treatment. The experiment of Ma et al. also proved this point of view. In the treatment without the application of fertilizer, NaOH-Po was the main source of Po [50]. NaOH-Po is mainly Po adsorbed on the surface of iron–aluminum complexes neutralized by humic acid, and it is a moderately labile Po. An increase in its content may be related to an increase in ALP activity.
According to the RDA analysis, Shinella, Ensifer and Bradyrhizobium were the main drivers of changes in soil P fractions, Enisifer is often reported to be a bacterium that promotes plant growth [51]. In this study, the abundance of Ensifer in the N1P0K1 and N0P0K0 treatments was 10.58 and 8 times that of N1P1K1, respectively, and Ensifer was significantly negatively correlated with labile P and moderately labile P. In other words, without the application of P fertilizer, Enisifer is highly induced to participate in the regulation of the soil P conversion process. This was similar to the results obtained by Zhuo et al. who found that Ensifer was an IPB that promotes the conversion from redox-sensitive P (BD-P) into NaOH-P, and the presence of rhizobium increases the content of NaOH-P that is easily exchanged at the solid–liquid interface [52]. Surprisingly, it was found that Ensifer also had a greater ability to dissolve Pi. Therefore, after treatment without P fertilizer application, Ensifer simultaneously mineralized Po and dissolved Pi to improve soil P availability. This may have also been related to the fact that the test soil for this experiment was calcareous soil. Calcareous soil is mainly composed of insoluble calcium phosphate, and Ensifer has a high solubilization ability to dissolve for calcium phosphate [53]. Yu et al. also found that the content of N-fixing symbionts in rice soil increased after long-term planting, which promoted an increase in NaOH-Po content. Previous studies showed that Shinella is involved in the transformation process of P fractions, and it is rich in the gcd gene encoding the glucose dehydrogenase of cheninoa protein, which plays a key role in the metabolism of dissolved Pi [54]. This better explained why the abundance of Shinella in the N1P1K1 treatment was significantly higher than that in the N1P0K1 and N0P0K0 treatments. Continuous application of P fertilizer would significantly increase the soil Pi content and, thus, increase the Shinella abundance.

4.3. Characteristics of the Variations in Cucumber Yield and P Uptake and the Relationship with Soil P Fractions

As a regulator of plant metabolism, P is involved in the metabolism of proteins, fats, and carbohydrates in crops, and it plays an important role in plant growth, development, and yield formation [55]. Studies have shown that the proper accumulation of soil P effectively enhances the level of P supply and increases the crop yield, but excessive P dose not promote higher crop yields or improved quality, instead increasing the risk of P loss [56]. The P requirement of vegetable crops is relatively high, and the threshold of AP in vegetable soils in China is 58.0 mg·kg−1 [57]. In this study, in greenhouse soil with a high basal AP content (about 156.3 mg·kg−1), it was found that four consecutive crops without P fertilization did not affect cucumber yields, and the yields were still higher than those in the continuous P fertilization treatment (Figure 1). This is consistent with the results of previous studies. Reducing the annual P fertilizer input by 33% in wheat–maize rotation soil for four consecutive crops did not reduce yield [58]. However, studies showed that there was no significant change in maize yield for five years of continuous reduction in P fertilizer application, and the yield without P fertilizer application began to decrease in the fourth year of the experiment. This was inconsistent with the results of this study. The reason may be that the soil AP in this study was 121.92 mg·kg−1 after four consecutive cropping rounds without P fertilizer, which was much higher than that in maize soil (where the AP was about 19 mg·kg−1), indicating a greater potential for P reduction in the GVP system. The yield of the N0P0K0 treatment was notably lower than that in the N1P0K1 treatment, which indicated that the application of N fertilizer had a significant effect on increasing the yield. Although there was no difference in the TN content between the two treatments, the AN in the N0P0K0 treatment was significantly lower than that in the N1P0K0 treatment, so the yield in the N0P0K0 treatment was lower.
Furthermore, the VPA revealed that the contribution rate of P fractions to cucumber yield was higher. The MSE model indicated that NaHCO3-Pi, NaOH-Pi, and Conc. HCl-Po contributed the most to the yield. This was consistent with the findings of Nziguheba et al. in the maize soil of Kenya [59]. Soil NaHCO3-Pi and NaOH-Pi were the main sources of P for crops, and they contributed more to maintaining high crop yields. These results indicated that NaHCO3-Pi and NaOH-Pi were important sources of P in plants in the short term.

5. Conclusions

This study found that solar greenhouses with P-rich soil have great potential for reduced P fertilizer application during vegetable production. Reduced application of P fertilizer in combination with other fertilizers will inhibit crop growth and reduce crop yield. However, reducing only P fertilizer for four consecutive crops could significantly improve the P surplus, maintain a cucumber yield equivalent to that in a conventional P fertilizer treatment, and increase the proportion of P distribution in the fruit. Following the reduction of P fertilizer application, labile Pi (Resin-Pi) emerged as the primary P source available to cucumber plants. Notably, the level of moderately labile Pi/TPi was significantly higher than that in the treatment with continuous P fertilizer application. Reducing the P fertilizer input significantly increased the soil ALP activity, the absolute abundance of the phoD gene, and the relative abundance of Ensifer within the associated microbial communities, forming a more stable and efficient network structure of key P-enhancing bacterial communities. The mineralization of moderately labile Pi to moderately labile Po was promoted. Our study further revealed that N-fixing symbiotic bacteria play a pivotal role in modulating phosphorus fractions, serving as the primary factor sustaining high crop yields. In light of these findings, future research endeavors should prioritize the development of P enhancers tailored specifically for N-fixing symbiotic bacteria in greenhouse settings characterized by P-rich soils, with the aim of conserving P resources.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture14111919/s1, Table S1: Effects of P fertilizer reduction on P uptake and distribution of cucumbers in the solar greenhouse; Table S2: The relative distribution of Po under P fertilizer reduction; Table S3. The relative distribution of Pi under P fertilizer reduction; Table S4. Effects of P fertilizer reduction on the phylum level of phoD gene bacterial community; Table S5. Pearson correlation analysis of soil phoD bacterial dominant taxa and phosphorus fractions.

Author Contributions

Conceptualization, T.B.; Methodology, T.B. and Z.W.; Software, T.B. and X.S.; Validation, T.B. and S.W.; Formal analysis, T.B.; Investigation, T.B. and T.W.; Resources, T.B. and Z.S.; Data curation, T.B., X.S. and T.W.; Writing—original draft preparation, T.B.; Writing—review and editing, T.B and Z.S.; Visualization, T.B. and H.F.; Supervision, Z.S. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financially supported by the China Agriculture Research System (CARS-23).

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors have declared that no competing interests exist. The funders have no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  1. Yang, X.; Liu, C.; Liang, C.; Wang, T.; Tian, J. The Phosphorus-Iron Nexus: Decoding the Nutrients Interaction in Soil and Plant. Int. J. Mol. Sci. 2024, 25, 6992. [Google Scholar] [CrossRef]
  2. Li, J.; Wan, X.; Liu, X.; Chen, Y.; Slaughter, L.C.; Weindorf, D.C.; Dong, Y. Changes in soil physical and chemical characteristics in intensively cultivated greenhouse vegetable fields in North China. Soil Tillage Res. 2019, 195, 104366. [Google Scholar] [CrossRef]
  3. Xu, M.-Z.; Wang, Y.-H.; Nie, C.-E.; Song, G.-P.; Xin, S.-N.; Lu, Y.-L.; Bai, Y.-L.; Zhang, Y.-J.; Wang, L. Identifying the critical phosphorus balance for optimizing phosphorus input and regulating soil phosphorus effectiveness in a typical winter wheat–summer maize rotation system in North China. J. Integr. Agric. 2023, 22, 3769–3782. [Google Scholar] [CrossRef]
  4. Lan, Z.M.; Lin, X.J.; Wang, F.; Zhang, H.; Chen, C.R. Phosphorus availability and rice grain yield in a paddy soil in response to long-term fertilization. Biol. Fertil. Soils 2012, 48, 579–588. [Google Scholar] [CrossRef]
  5. Zhang, W.; Zhang, Y.; An, Y.; Chen, X. Phosphorus fractionation related to environmental risks resulting from intensive vegetable cropping and fertilization in a subtropical region. Environ. Pollut. 2021, 269, 116098. [Google Scholar] [CrossRef] [PubMed]
  6. Wang, Y.; Zhao, X.; Guo, Z.; Jia, Z.; Wang, S.; Ding, K. Response of soil microbes to a reduction in phosphorus fertilizer in rice-wheat rotation paddy soils with varying soil P levels. Soil Tillage Res. 2018, 181, 127–135. [Google Scholar] [CrossRef]
  7. Bai, Y.; Zhu, J.; Deng, B.; Shi, H.; Wang, Z.; Han, D.; Duan, J.; Wang, D. Short-Term Effect of the Addition of Rice Husk Gasification Slag on the Movement and Transformation of Phosphorus in Different Soil Types. Sustainability 2020, 12, 2458. [Google Scholar] [CrossRef]
  8. Wei, K.; Chen, Z.; Jiang, N.; Zhang, Y.; Feng, J.; Tian, J.; Chen, X.; Lou, C.; Chen, L. Effects of mineral phosphorus fertilizer reduction and maize straw incorporation on soil phosphorus availability, acid phosphatase activity, and maize grain yield in northeast China. Arch. Agron. Soil Sci. 2020, 67, 66–78. [Google Scholar] [CrossRef]
  9. Zhang, Y.; Gao, W.; Luan, H.; Tang, J.; Li, R.; Li, M.; Zhang, H.; Huang, S. Long-Term organic substitution management affects soil phosphorus speciation and reduces leaching in greenhouse vegetable production. J. Clean. Prod. 2021, 327, 129464. [Google Scholar] [CrossRef]
  10. Wu, Q.; Huang, Z.; Zhu, P.; Wang, B.; Zhang, S.; Ao, J. Long-term change in phosphorus behavior and the degree of P saturation in typical cropland after different fertilization practices. Soil Sci. Plant Nutr. 2021, 68, 202–214. [Google Scholar] [CrossRef]
  11. Schaap, K.J.; Fuchslueger, L.; Hoosbeek, M.R.; Hofhansl, F.; Martins, N.P.; Valverde-Barrantes, O.J.; Hartley, I.P.; Lugli, L.F.; Quesada, C.A. Litter inputs and phosphatase activity affect the temporal variability of organic phosphorus in a tropical forest soil in the Central Amazon. Plant Soil 2021, 469, 423–441. [Google Scholar] [CrossRef]
  12. Nishigaki, T.; Tsujimoto, Y.; Rinasoa, S.; Rakotoson, T.; Andriamananjara, A.; Razafimbelo, T. Phosphorus uptake of rice plants is affected by phosphorus forms and physicochemical properties of tropical weathered soils. Plant Soil 2018, 435, 27–38. [Google Scholar] [CrossRef]
  13. Gatiboni, L.; Djalma, E.S.; Tiecher, T.; Murilo, G.V.; Danilo, R.D.S.; João, K.; Gustavo, B. Plant uptake of legacy phosphorus from soils without P fertilization. Nutr. Cycl. Agroecosystems 2021, 119, 139–151. [Google Scholar] [CrossRef]
  14. Bahramnejad, A.R.; Abad, H.H.S.; Madani, H. Effect of Irrigation Regimes and Phosphorus Fertilization on Water-use Efficiency, Phosphorus-agronomic Efficiency and Yield of Grass Pea (Lathyrus sativus L.) Ecotypes. Legume Res. Int. J. 2021, 44, 1038–1045. [Google Scholar] [CrossRef]
  15. Ye, L.; Zhao, X.; Bao, E.; Li, J.S.; Zhou, Z.R.; Cao, K. Bio-Organic fertilizer with reduced rates of chemical fertilization improves soil fertility and enhances tomato yield and quality. Sci. Rep. 2020, 10, 177. [Google Scholar] [CrossRef]
  16. Ragot, S.A.; Kertesz, M.A.; Bünemann, E.K. Diversity of the phoD alkaline phosphatase gene in soil. Appl. Environ. Microbiol. 2015, 81, 7281. [Google Scholar] [CrossRef]
  17. Shi, W.; Xing, Y.; Zhu, Y.; Gao, N.; Ying, Y. Diverse responses of pqqC- and phoD-harbouring bacterial communities to variation in soil properties of Moso bamboo forests. Microb. Biotechnol. 2022, 15, 2097–2111. [Google Scholar] [CrossRef]
  18. Luo, G.; Ling, N.; Nannipieri, P.; Chen, H.; Raza, W.; Wang, M.; Guo, S.; Shen, Q. Long-Term fertilisation regimes affect the composition of the alkaline phosphomonoesterase encoding microbial community of a vertisol and its derivative soil fractions. Biol. Fertil. Soils 2017, 53, 375–388. [Google Scholar] [CrossRef]
  19. Chen, X.; Jiang, N.; Condron, L.M.; Dunfield, K.E.; Chen, Z.; Wang, J.; Chen, L. Impact of long-term phosphorus fertilizer inputs on bacterial phoD gene community in a maize field, Northeast China. Sci. Total Environ. 2019, 669, 1011–1018. [Google Scholar] [CrossRef]
  20. Gou, X.; Ren, Y.; Qin, X.; Wei, X.; Wang, J. Global patterns of soil phosphatase responses to nitrogen and phosphorus fertilization. Pedosphere 2024, 34, 200–210. [Google Scholar] [CrossRef]
  21. Bi, Q.F.; Li, K.J.; Zheng, B.X.; Liu, X.P.; Li, H.Z.; Jin, B.J.; Ding, K.; Yang, X.R.; Lin, X.Y.; Zhu, Y.G. Partial replacement of inorganic phosphorus (P) by organic manure reshapes phosphate mobilizing bacterial community and promotes P bioavailability in a paddy soil. Sci. Total Environ. 2020, 703, 134977. [Google Scholar] [CrossRef] [PubMed]
  22. Yu, X.Z.; Chen, Q.; Shi, W.C.; Sun, X.; Dong, J.J.; Li, J.; Wang, H.T.; Gao, J.G.; Liu, Z.G.; Zhang, M. Interactions between phosphorus availability and microbes in a wheat–maize double cropping system: A reduced fertilization scheme. J. Integr. Agric. 2022, 21, 840–854. [Google Scholar] [CrossRef]
  23. Roohi, M.; Arif, M.S.; Yasmeen, T.; Bragazza, L. Effects of cropping system and fertilization regime on soil phosphorous are mediated by rhizosphere-microbial processes in a semi-arid agroecosystem. J. Environ. Manag. 2020, 271, 111033. [Google Scholar] [CrossRef] [PubMed]
  24. Yu, X.; Tong, D.; Wang, S.; Wang, D.; Xu, L.; Li, B.; Sun, W.; Wang, Y. Long-Term rice cultivation promoted microbial mineralization of organic P in a black soil. Soil Sci. Soc. Am. J. 2022, 86, 540–551. [Google Scholar] [CrossRef]
  25. Subramanyam, R.; Meng, X.; Chen, W.-W.; Wang, Y.-Y.; Huang, Z.-R.; Ye, X.; Chen, L.-S.; Yang, L.-T. Effects of phosphorus deficiency on the absorption of mineral nutrients, photosynthetic system performance and antioxidant metabolism in Citrus grandis. PLoS ONE 2021, 16, e0246944. [Google Scholar] [CrossRef]
  26. Bao, S.D. Soil and Agricultural Chemistry Analysis, 3rd ed.; China Agriculture Press: Beijing, China, 2000. [Google Scholar]
  27. Tiessen, H.; Moir, J.O. Characterization of available P by sequential extraction. Soil Sampl. Methods Anal. 1993, 7, 5–229. [Google Scholar]
  28. Hedley, M.J.; Stewart, J.W.B.; Chauhan, B.S. Changes in Inorganic and Organic Soil Phosphorus Fractions Induced by Cultivation Practices and by Laboratory Incubations. Soil Sci. Soc. Am. J. 1982, 46, 970–976. [Google Scholar] [CrossRef]
  29. Tabatabai, M. Methods of soil analysis: Part 2—Microbiological and biochemical properties. Soil Enzym. 1994, 5, 775–833. [Google Scholar]
  30. Ragot, S.A.; Huguenin-Elie, O.; Kertesz, M.A.; Frossard, E.; Bünemann, E.K. Total and active microbial communities and phoD as affected by phosphate depletion and pH in soil. Plant Soil 2016, 408, 15–30. [Google Scholar] [CrossRef]
  31. Ragot, S.A. Development and Application of Molecular Tools to Investigate Microbial Alkaline Phosphatase Genes in Soil. Ph.D. Thesis, ETH Zurich, Zurich, Switzerland, 2016. [Google Scholar]
  32. Wang, Y.; Huang, R.; Xu, G.; Li, J.; Wang, Z.; Ci, E.; Gao, M. Soil alkaline phosphatase activity and bacterial phoD gene abundance and diversity under regimes of inorganic fertilizer reduction with straw. J. Soils Sediments 2020, 21, 388–402. [Google Scholar] [CrossRef]
  33. Fraser, T.D.; Lynch, D.H.; Bent, E.; Entz, M.H.; Dunfield, K.E. Soil bacterial phoD gene abundance and expression in response to applied phosphorus and long-term management. Soil Biol. Biochem. 2015, 88, 137–147. [Google Scholar] [CrossRef]
  34. Yang, Y.; Liu, L.; Liu, X.; Ma, Q.; Liu, J. Impact of Maize Growth Stage on Soil Bacterial Communities and Functions in Dryland Under P Fertilization: A Comprehensive Analysis. J. Soil Sci. Plant Nutr. 2024, 24, 1172–1182. [Google Scholar] [CrossRef]
  35. Wang, M.; Wu, Y.; Zhao, J.; Liu, Y.; Chen, Z.; Tang, Z.; Tian, W.; Xi, Y.; Zhang, J. Long-term fertilization lowers the alkaline phosphatase activity by impacting the phoD-harboring bacterial community in rice-winter wheat rotation system. Sci. Total Environ. 2022, 821, 153406. [Google Scholar] [CrossRef] [PubMed]
  36. Hu, Y.; Xia, Y.; Sun, Q.; Liu, K.; Chen, X.; Ge, T.; Zhu, B.; Zhu, Z.; Zhang, Z.; Su, Y. Effects of long-term fertilization on phoD-harboring bacterial community in Karst soils. Sci. Total Environ. 2018, 628, 53–63. [Google Scholar] [CrossRef]
  37. Lang, M.; Zou, W.; Chen, X.; Zou, C.; Zhang, W.; Deng, Y.; Zhu, F.; Yu, P.; Chen, X. Soil Microbial Composition and phoD Gene Abundance Are Sensitive to Phosphorus Level in a Long-Term Wheat-Maize Crop System. Front. Microbiol. 2021, 11, 605955. [Google Scholar] [CrossRef]
  38. Long, X.-E.; Yao, H.; Huang, Y.; Wei, W.; Zhu, Y.-G. Phosphate levels influence the utilisation of rice rhizodeposition carbon and the phosphate-solubilising microbial community in a paddy soil. Soil Biol. Biochem. 2018, 118, 103–114. [Google Scholar] [CrossRef]
  39. Xin, Y.-Y.; Rahman, A.; Li, H.-X.; Xu, T.; Ding, G.-C.; Li, J. Modification of total and phosphorus mineralizing bacterial communities associated with Zea mays L. through plant development and fertilization regimes. J. Integr. Agric. 2021, 20, 3026–3038. [Google Scholar] [CrossRef]
  40. Khan, A.; Zhang, G.; Li, T.; He, B. Fertilization and cultivation management promotes soil phosphorus availability by enhancing soil P-cycling enzymes and the phosphatase encoding genes in bulk and rhizosphere soil of a maize crop in sloping cropland. Ecotoxicol. Environ. Saf. 2023, 264, 115441. [Google Scholar] [CrossRef]
  41. Zheng, M.M.; Wang, C.; Li, W.X.; Guo, L.; Cai, Z.J.; Wang, B.R.; Chen, J.; Shen, R.F. Changes of acid and alkaline phosphatase activities in long-term chemical fertilization are driven by the similar soil properties and associated microbial community composition in acidic soil. Eur. J. Soil Biol. 2021, 104, 103312. [Google Scholar] [CrossRef]
  42. Nunes, R.d.S.; de Sousa, D.M.G.; Goedert, W.J.; de Oliveira, L.E.Z.; Pavinato, P.S.; Pinheiro, T.D. Distribution of Soil Phosphorus Fractions as a Function of Long-Term Soil Tillage and Phosphate Fertilization Management. Front. Earth Sci. 2020, 8, 350. [Google Scholar] [CrossRef]
  43. Kumar, Y.; Naresh, R.K.; Dhaliwal, S.S.; Sharma, V.; Kumar, R.; Mandal, A. Impact of NPK Enriched Bio-Compost on Rice Yield and Sustainability of Nutrients in Sandy Loam Soils of India. Commun. Soil Sci. Plant Anal. 2022, 53, 2996–3007. [Google Scholar] [CrossRef]
  44. Sun, F.; Sun, N.; Ma, X.; Zhou, B.; Zhu, P.; Gao, H.; Xu, M. The Application of Fertilizer Phosphorus Affected Olsen P and the Phosphorus Fractions of Hedley Method in Black Soil. Agronomy 2022, 12, 3146. [Google Scholar] [CrossRef]
  45. Pavinato, P.S.; Gotz, L.F.; Teles, A.P.B.; Arruda, B.; Herrera, W.B.; Chadwick, D.R.; Jones, D.L.; Withers, P.J.A. Legacy soil phosphorus bioavailability in tropical and temperate soils: Implications for sustainable crop production. Soil Tillage Res. 2024, 244, 106228. [Google Scholar] [CrossRef]
  46. Bibi, S.; Irshad, M.; Mohiuddin, M.; Sher, S.; Tariq, M.A.U.R.; Ng, A.W.M. Distribution of Phosphorus Fractions in Orchard Soils in Relation to Soil Properties and Foliar P Contents. Sustainability 2022, 14, 3966. [Google Scholar] [CrossRef]
  47. Wang, Q.; Zhang, N.; Chen, Y.; Qin, Z.; Jin, Y.; Zhu, P.; Peng, C.; Colinet, G.; Zhang, S.; Liu, J. The Phosphorus Availability in Mollisol Is Determined by Inorganic Phosphorus Fraction under Long-Term Different Phosphorus Fertilization Regimes. Agronomy 2022, 12, 2364. [Google Scholar] [CrossRef]
  48. Li, Y.; Xu, Z.; Zhang, L.; Chen, W.; Feng, G. Dynamics between soil fixation of fertilizer phosphorus and biological phosphorus mobilization determine the phosphorus budgets in agroecosystems. Agric. Ecosyst. Environ. 2024, 375, 109174. [Google Scholar] [CrossRef]
  49. Guppy, C. Is soil phosphorus fractionation as valuable as we think? Plant Soil 2021, 459, 19–21. [Google Scholar] [CrossRef]
  50. Mahmood, M.; Tian, Y.; Ma, Q.; Ahmed, W.; Mehmood, S.; Hui, X.; Wang, Z. Changes in Phosphorus Fractions and Its Availability Status in Relation to Long Term P Fertilization in Loess Plateau of China. Agronomy 2020, 10, 1818. [Google Scholar] [CrossRef]
  51. Huang, B.; Yan, D.; Ouyang, C.; Zhang, D.; Zhu, J.; Liu, J.; Li, Y.; Wang, Q.; Han, Q.; Cao, A. Chloropicrin fumigation alters the soil phosphorus and the composition of the encoding alkaline phosphatase PhoD gene microbial community. Sci. Total Environ. 2020, 711, 135080. [Google Scholar] [CrossRef]
  52. Zhuo, T.Y.; Ding, Y.; Wan, Q.; Li, S.M.; Chai, B.B.; Lei, X.H. Effects of hydrostatic pressure on phosphorus transformation at the water-sediment interface of a deep reservoir:novel insights into bacterial community and functional genes. J. Soils Sediments 2021, 21, 3367–3379. [Google Scholar] [CrossRef]
  53. Li, K.S.; Van Zeghbroeck, J.; Liu, Q.C.; Zhang, S.A. Isolating and Characterizing Phosphorus Solubilizing Bacteria from Rhizospheres of Native Plants Grown in Calcareous Soils. Front. Environ. Sci. 2021, 9, 802563. [Google Scholar] [CrossRef]
  54. Chen, W.; Zhan, Y.; Zhang, X.; Shi, X.; Wang, Z.; Xu, S.; Chang, Y.; Ding, G.; Li, J.; Wei, Y. Influence of carbon-to-phosphorus ratios on phosphorus fractions transformation and bacterial community succession in phosphorus-enriched composting. Bioresour. Technol. 2022, 362, 127786. [Google Scholar] [CrossRef] [PubMed]
  55. Maity, A.; Marathe, R.A.; Sarkar, A.; Basak, B.B. Phosphorus and potassium supplementing bio-mineral fertilizer augments soil fertility and improves fruit yield and quality of pomegranate. Sci. Hortic. 2022, 303, 111234. [Google Scholar] [CrossRef]
  56. Bai, S.; Tan, J.; Zhang, Z.; Wei, M.; Zhang, H.; Jiang, X. Phosphorus speciation and colloidal phosphorus responses to short-term cessation of fertilization in a lime concretion black soil. Pedosphere 2023, 33, 948–959. [Google Scholar] [CrossRef]
  57. Wang, Y.L.; Zhang, H.L.; Tang, J.W.; Xu, J.B.; Kou, T.J.; Huang, H.M. Accelerated phosphorus accumulation and acidification of soils under plastic greenhouse condition in four representative organic vegetable cultivation sites. Sci. Hortic. 2015, 195, 67–73. [Google Scholar] [CrossRef]
  58. Xiao, L.; Chen, G.L.; Wang, H.; Li, Y.X.; Li, C.; Cheng, L.; Wu, W.E.; Xiao, X.; Zhu, Y.Y. Improvement of P Use Efficiency and P Balance of Rice-Wheat Rotation System According to the Long-Term Field Experiments in the Taihu Lake Basin. Front. Environ. Sci. 2022, 10, 932833. [Google Scholar] [CrossRef]
  59. Nziguheba, G.; Merckx, R.; Palm, C.A. Soil phosphorus dynamics and maize response to different rates of phosphorus fertilizer applied to an Acrisol in western Kenya. Plant Soil 2002, 243, 1–10. [Google Scholar] [CrossRef]
Agriculture 14 01919 g001
Figure 1. Effects of P fertilizer reduction on plant height, biomass, yield and plant P uptake of cucumber in solar greenhouse. Different lower-case letters indicate significant differences between samples (p < 0.05).
Figure 1. Effects of P fertilizer reduction on plant height, biomass, yield and plant P uptake of cucumber in solar greenhouse. Different lower-case letters indicate significant differences between samples (p < 0.05).
Agriculture 14 01919 g001
Agriculture 14 01919 g002
Figure 2. Effects of P reduction on soil P fractions of cucumber in solar greenhouse. Different lower-case letters indicate significant differences between samples (p < 0.05).
Figure 2. Effects of P reduction on soil P fractions of cucumber in solar greenhouse. Different lower-case letters indicate significant differences between samples (p < 0.05).
Agriculture 14 01919 g002
Agriculture 14 01919 g003
Figure 3. Effects of P fertilizer reduction on ACP, ALP and PDE activity of cucumber in solar greenhouse. ALP alkaline phosphomonoesterase, ACP acid phosphomonoesterase, PDE phosphodiesterase. Different lower-case letters indicate significant differences between samples (p < 0.05).
Figure 3. Effects of P fertilizer reduction on ACP, ALP and PDE activity of cucumber in solar greenhouse. ALP alkaline phosphomonoesterase, ACP acid phosphomonoesterase, PDE phosphodiesterase. Different lower-case letters indicate significant differences between samples (p < 0.05).
Agriculture 14 01919 g003
Agriculture 14 01919 g004
Figure 4. Effects of P fertilizer reduction on bar chart of the abundance of phoD (a), and the Shannon diversity index for phoD (b) of cucumber in solar greenhouse, the principal component analysis (PCA) of phoD-harboring bacterial communities (c). Different lower-case letters indicate significant differences between samples (p < 0.05).
Figure 4. Effects of P fertilizer reduction on bar chart of the abundance of phoD (a), and the Shannon diversity index for phoD (b) of cucumber in solar greenhouse, the principal component analysis (PCA) of phoD-harboring bacterial communities (c). Different lower-case letters indicate significant differences between samples (p < 0.05).
Agriculture 14 01919 g004
Agriculture 14 01919 g005
Figure 5. Heatmap of phoD gene communities at the genus level. Only the relative abundance of the genus in samples > 1% was shown.
Figure 5. Heatmap of phoD gene communities at the genus level. Only the relative abundance of the genus in samples > 1% was shown.
Agriculture 14 01919 g005
Agriculture 14 01919 g006
Figure 6. Analysis of significant differences between groups on the dominant genus of the phoD gene bacterial community under P fertilizer reduction (one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001).
Figure 6. Analysis of significant differences between groups on the dominant genus of the phoD gene bacterial community under P fertilizer reduction (one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001).
Agriculture 14 01919 g006
Agriculture 14 01919 g007
Figure 7. Network of bacteria depending on P fertilizer reduction, analysis based on random matrix theory (RMT) from OTU profiles. The size of each node is in proportion to the number of connections. The OTUs acting as generalists are labeled in the networks.
Figure 7. Network of bacteria depending on P fertilizer reduction, analysis based on random matrix theory (RMT) from OTU profiles. The size of each node is in proportion to the number of connections. The OTUs acting as generalists are labeled in the networks.
Agriculture 14 01919 g007
Agriculture 14 01919 g008
Figure 8. RDA plot displays the interrelationships between environmental factors and various the dominant genus of the phoD gene in the N1P1K1 and N1P0K1 treatments (a), and in the N1P1K1 versus N0P0K0 treatments (b).
Figure 8. RDA plot displays the interrelationships between environmental factors and various the dominant genus of the phoD gene in the N1P1K1 and N1P0K1 treatments (a), and in the N1P1K1 versus N0P0K0 treatments (b).
Agriculture 14 01919 g008
Agriculture 14 01919 g009
Figure 9. RDA plot displays the interrelationships between the dominant genus of the phoD gene and various soil P fractions in the N1P1K1 and N1P0K1 treatments (a), and in the N1P1K1 versus N0P0K0 treatments (b).
Figure 9. RDA plot displays the interrelationships between the dominant genus of the phoD gene and various soil P fractions in the N1P1K1 and N1P0K1 treatments (a), and in the N1P1K1 versus N0P0K0 treatments (b).
Agriculture 14 01919 g009
Agriculture 14 01919 g010
Figure 10. The VPA plot exhibits the correlation between environmental variables and P fractions, plant height, yield, and P uptake (a). Random forest model analysis conducts to pinpoint the primary predictors that significantly influence yield (b).
Figure 10. The VPA plot exhibits the correlation between environmental variables and P fractions, plant height, yield, and P uptake (a). Random forest model analysis conducts to pinpoint the primary predictors that significantly influence yield (b).
Agriculture 14 01919 g010
Table 1. Basic properties and P fraction content of the tested soil.
Table 1. Basic properties and P fraction content of the tested soil.
MaterialspHTCTNTPTKSOMANAPAK
g·kg−1g·kg−1g·kg−1g·kg−1g·kg−1mg·kg−1mg·kg−1mg·kg−1
Original soil7.3021.362.090.9123.7236.8240.30156.30231.30
Resin-PNaHCO3-PiNaOH-PiDil. HCl-PiConc. HCl-PiNaHCO3-PoNaOH-PoConc. HCl-PoResidual-P
mg·kg−1mg·kg−1mg·kg−1mg·kg−1mg·kg−1mg·kg−1mg·kg−1mg·kg−1mg·kg−1
185.10142.10130.70208.70130.1029.2081.3017.8057.20
Note: TC soil total C, TN soil total N, TP soil total P, TK soil total K, SOM soil organic matter, AN soil available N, AP soil available P, AK soil available K.
Table 2. Effects of P fertilizer reduction on soil chemical properties of cucumber in solar greenhouse.
Table 2. Effects of P fertilizer reduction on soil chemical properties of cucumber in solar greenhouse.
TreatmentN1P1K1N1P0K1N0P0K0
pH7.15 ± 0.03 a7.26 ± 0.05 a7.27 ± 0.03 a
TC (g·kg−1)21.73 ± 0.05 ab20.51 ± 0.37 b22.43 ± 0.52 a
TN (g·kg−1)2.19 ± 0.04 a2.01 ± 0.05 b2.05 ± 0.02 b
TP (g·kg−1)1.05 ± 0.05 a0.83 ± 0.03 b0.77 ± 0.01 b
TK (g·kg−1)23.94 ± 0.35 a23.38 ± 1.64 a23.70 ± 0.69 a
SOM (g·kg−1)36.23 ± 3.18 a33.71 ± 1.59 a37.60 ± 0.61 a
AN (mg·kg−1)41.61 ± 1.08 ab43.36 ± 0.70 a38.5 ± 1.01 b
AP (mg·kg−1)233.37 ± 17.03 a121.92 ± 5.87 b113.45 ± 5.09 b
AK (mg·kg−1)677.92 ± 24.23 a587.32 ± 11.21 b129.59 ± 12.69 c
M3-Ca (mg·kg−1)1073.67 ± 12.49 c1110.67 ± 60.92 b1383.33 ± 62.81 a
M3-Al (mg·kg−1)262.67 ± 18.67 a286.67 ± 13.86 a309.67 ± 7.45 a
M3-Fe (mg·kg−1)100.67 ± 6.89 b113.67 ± 0.33 ab118.00 ± 3.06 a
Note: M3-Ca, M3-Mg, M3-Fe and M3-Al are Mehlich-3 extractable Ca, Mg, Fe and Al, respectively. Mean ± Standard error (n = 3). Different lower-case letters in the same column indicate significant differences between samples (p < 0.05).
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.

Share and Cite

MDPI and ACS Style

Bian, T.; Wang, Z.; Wang, S.; Shan, X.; Wang, T.; Fu, H.; Sun, Z. Effect of P Reduction on phoD-Harboring Bacteria Community in Solar Greenhouse Soil. Agriculture 2024, 14, 1919. https://doi.org/10.3390/agriculture14111919

AMA Style

Bian T, Wang Z, Wang S, Shan X, Wang T, Fu H, Sun Z. Effect of P Reduction on phoD-Harboring Bacteria Community in Solar Greenhouse Soil. Agriculture. 2024; 14(11):1919. https://doi.org/10.3390/agriculture14111919

Chicago/Turabian Style

Bian, Ting, Zhen Wang, Shuang Wang, Xuan Shan, Tianqi Wang, Hongdan Fu, and Zhouping Sun. 2024. "Effect of P Reduction on phoD-Harboring Bacteria Community in Solar Greenhouse Soil" Agriculture 14, no. 11: 1919. https://doi.org/10.3390/agriculture14111919

APA Style

Bian, T., Wang, Z., Wang, S., Shan, X., Wang, T., Fu, H., & Sun, Z. (2024). Effect of P Reduction on phoD-Harboring Bacteria Community in Solar Greenhouse Soil. Agriculture, 14(11), 1919. https://doi.org/10.3390/agriculture14111919

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop