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Article

Effects of Exogenous Application of Phenolic Acid on Soil Nutrient Availability, Enzyme Activities, and Microbial Communities

by
Yi Zhou
1,
Yihang Liu
1,
Chaoqiang Jiang
2,
Zeinab El-Desouki
3,
Muhammad Riaz
4,
Chenlu Wang
1,
Xueping Zhang
1,
Jiayi Ding
1,
Zhenghao Chen
1,
Huaiwei Liu
5,
Jia Shen
2 and
Hao Xia
2,*
1
College of Resources and Environment, Anhui Science and Technology University, Chuzhou 233100, China
2
Institute of Industrial Crops, Anhui Academy of Agricultural Sciences, Hefei 230001, China
3
Microelement Research Center, College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, China
4
College of Resources and Environment, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China
5
Agriculture and Rural Bureau of Dingtao District, Heze 274100, China
*
Author to whom correspondence should be addressed.
Agriculture 2025, 15(10), 1067; https://doi.org/10.3390/agriculture15101067
Submission received: 7 April 2025 / Revised: 25 April 2025 / Accepted: 14 May 2025 / Published: 15 May 2025
(This article belongs to the Section Agricultural Soils)
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Abstract

:
Phenolic acids are important allelochemicals that contribute to obstacles in continuous cropping systems, significantly impacting soil nutrients, enzyme activities, and the composition of microbial communities. This study explored the effects of treatment time and the concentration of various phenolic acids (salicylic acid and p-hydroxybenzoic acid) on soil nutrients, enzyme activity, and soil microorganisms through cultivation experiments. The results indicated that high-concentration phenolic acid treatment negatively affected the availability of soil nutrients by acidifying the soil, as reflected in the low soil pH, compared to the untreated control. Moreover, the soil extracellular enzymes exhibited varying degrees of improvement when phenolic acids were added. Multi-element analysis revealed that treatment duration, concentration, and the type of phenolic acid significantly affected soil nutrient levels and enzyme activity. Additionally, structural equation modeling indicated a significant correlation between the concentration of phenolic acids and the diversity of microorganisms. Phenolic acids influence the soil ecological environment by altering the relative abundance of functional microorganisms (p_Patescibacteria and p_Mortierellomycota) in the soil. Thus, comprehensive regulation and control of continuous cropping obstacles can be achieved by adjusting the micro-ecological environment of the soil, which, in turn, affects phenolic acid substances present in the soil, thereby alleviating continuous cropping obstacles.

1. Introduction

The rhizosphere refers to the micro-soil region adjacent to plant roots and is distinguished from surrounding soil by its unique physical, chemical, and biological properties [1]. This area serves as a dynamic interface where material exchange occurs frequently within the plant–soil ecosystem, and it is characterized by high microbial activity [2]. Alterations in the composition of the microbial community within the rhizosphere significantly affect the release of plant root exudates, the circulation of soil materials, energy flow, and information transmission [3]. Root exudates refer to the organic substances released by living plants into the rhizosphere environment through various parts of the root system under specific conditions and can originate from two sources: root exudation and the decomposition of plant residues [4,5,6]. Root exudates are essential for managing the ecosystem of rhizosphere microorganisms, exerting a selective influence on the composition of the rhizosphere microbial community [7]. These exudates, which include sugars, amino acids, phenolic acids, and organic acids, are important for controlling the types and activities of microorganisms in the rhizosphere [8]. Therefore, studying the impact of these secretions on microbial communities can enhance the efficiency of nutrient resource utilization in crops, improve crop productivity, and promote sustainable agricultural development.
With the rise in agricultural intensification and the multiple cropping index, outbreaks of soil-borne pests and diseases, as well as challenges related to continuous cropping, are becoming more serious [9,10]. Research shows that rhizosphere microorganisms play a crucial role in assisting plants to resist the invasion of soil-borne pathogens [11]. Moreover, plant roots specifically adjust the composition and quantity of root exudates and recruit beneficial microorganisms that can utilize these specialized substances to combat soil pathogenic bacteria [5]. However, some root exudates can negatively impact the environment by inhibiting the abundance or function of beneficial microorganisms [12]. This negative effect is primarily manifested in three aspects: (1) root exudates facilitate the survival of soil-borne pathogens within the soil, as well as their migration and proliferation to the rhizosphere [13]; (2) root exudates disrupt beneficial microbial communities in the rhizosphere [14]; (3) root exudates compromise the root defense system [15].
Among various root exudates, phenolic acids are significant allelopathic substances, and their allelopathic mechanisms should be further investigated. The accumulation of allelopathic substances, such as saponins from P. notoginseng, p-hydroxybenzoic acid from peanuts, and cinnamic acid from tobacco, significantly reduces crop yield and quality [16]. These substances also promote the reproduction of pathogenic microorganisms, including Fusarium from P. notoginseng, Burkholderia from peanuts, and Ralstonia solanacearum from tobacco, thereby exacerbating the challenges of continuous cropping obstacles [17]. For instance, tobacco roots frequently secrete autotoxic substances, including p-hydroxybenzoic acid, myristic acid, and fumaric acid, which can enhance the growth of a soil-borne pathogen (Ralstonia solanacearum) [18]. Similarly, watermelon roots can secrete ferulic acid and salicylic acid, which can stimulate the proliferation of Fusarium oxysporum [19]. In leguminous crops, roots secrete benzoic acid, salicylic acid, and other substances that significantly inhibit the growth of Pseudomonas [20]. In addition, cucumber roots can secrete ferulic acid, which inhibits the growth of cucumber seedlings, decreases bacterial diversity, and enhances fungal diversity [21]. Understanding the role of root exudates in the composition of soil microbial communities is critical for developing strategies to mitigate soil-borne diseases and improve agricultural sustainability.
In this study, we conducted a pot experiment to explore the effects of root exudates (p-hydroxybenzoic acid; salicylic acid) on soil fertility, enzyme activity, and microbial community composition. Using high-throughput sequencing, we analyzed how phenolic acid (types, concentration, and processing time) impact the soil microbial community. The specific hypotheses of this study were as follows: (1) Phenolic acids can cause soil acidification and affect the effectiveness of available nutrients. (2) Phenolic acids exhibit a phenomenon characterized by the “low promotion and high suppression” of soil enzyme activity and microbial diversity. (3) Phenolic acids can influence rhizosphere microbial communities and reduce the abundance of beneficial microbial groups. The specific objective of this study was to elucidate the microbial mechanisms through which root exudates from continuous cropping exacerbate soil biological barriers.

2. Materials and Methods

2.1. Experimental Design and Site Description

The pot-based experiment was carried out in a greenhouse at Anhui Academy of Agricultural Sciences, Hefei City, Anhui Province, China (31°49′ N, 117°13′ E). The experimental soil was collected from topsoil (0–20 cm) in Xuancheng City, Anhui Province, China (30°56′ N, 118°45′ E). The basic soil properties included a pH of 5.38, available nitrogen (AN) at 60.71 mg/kg, available potassium (AK) at 156.72 mg/kg, available phosphorus (AP) at 41.35 mg/kg, and organic matter (SOM) at 19.85 g/kg.
We set 13 treatments, and the experiment was a 2 × 3 × 2 factorial experiment replicated 3 times in a completely randomized block design. The treatments included 3 factors: the phenolic acid substance (salicylic acid, S; p-hydroxybenzoic acid, P), phenolic acid concentration (0.5 mg/g, L; 1 mg/g, M; 2 mg/g, H), and processing time (10 d; 20 d). The control treatment (CK) was cultivated in deionized water. The rate of application of fertilizers to the soil was 0.480 g of urea, 0.264 g of P2O5, and 0.420 g of K2O. A total of 0.2 kg of soil was thoroughly mixed with fertilizers and subsequently placed in a plastic pot with a height of 15 cm and a diameter of 12 cm. During the experimental period, soil moisture was kept at 75% of field capacity using the gravimetric method. The pot experiment took place in a natural greenhouse and continued for 20 days.

2.2. Sampling and Analyses

Following the cultivation phase, soil samples were collected and divided into three separate sections. The first portion was air-dried to assess fundamental soil properties. The second portion was kept at 4 °C for the evaluation of soil extracellular enzymes, while the third portion was stored at −80 °C for microbial analysis. Soil pH was measured using a pH meter with a water-to-soil ratio of 2.5:1. Soil organic matter (SOM) content was determined using the potassium dichromate oxidation method with external heating. Alkaline hydrolyzable nitrogen (AN) was assessed through the alkaline hydrolysis diffusion boric acid absorption method. Available phosphorus (AP) was determined utilizing the colorimetric technique involving molybdenum and antimony. The levels of available potassium were measured by NH4OAc extraction followed by flame photometry (AP1200, Aopu Analytical Instrument Co., Ltd., Shanghai, China) according to Bao, 2000 [22]. Soil enzyme activity was assessed using the microplate reader method [23]. The procedure was as follows: A total of 2 g of fresh soil was weighed and placed into a 250 mL plastic bottle, followed by the addition of 200 mL of deionized water. The mixture was shaken for 40 min and then incubated in the dark at 25 °C for 4 h. After incubation, fluorescence intensity was measured using a microplate reader (Tecan, spark 10, Manne Dorf, Switzerland) at excitation and emission wavelengths of 365 nm and 450 nm, respectively [24].

2.3. Soil Microorganism Analysis

Soil microorganisms: Total microbial DNA was isolated using the EZNA® Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA). DNA quality was evaluated through 1% agarose gel electrophoresis, while its concentration and purity were measured with a NanoDrop 2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The primers of bacteria and fungi were 515F-806R in the V3-V4 region and ITS1F-ITS2R in the ITS region, respectively. The PCR products were recovered using a 2% agarose gel. The recovered products were then purified with the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and subsequently analyzed by 2% agarose gel electrophoresis. The detection and quantification of the purified products were carried out using a Quantus™ Fluorometer (Promega, Madison, WI, USA). Library construction was performed using the NEXTFLEX Rapid DNA-Seq Kit (Bioo Scientific Corp, Austin, TX, USA). Sequencing was conducted on Illumina’s MiSeq PE300 platform (Shanghai Majorbio Biopharm Technology Co., Ltd., Shanghai, China).
Fastp software (http://ccb.jhu.edu/software/FLASH/) was employed for the quality control of the original sequencing data, while Flash software was utilized for sequence splicing. The sequences were clustered into OTUs with Uparse software (Uparse v7.0.1001, http://drive5.com/uparse/), and chimeric sequences were removed based on a 97% similarity threshold. The RDP classifier, referencing the Silva 16S rRNA database (bacterium) and unite (fungus), was used to annotate the species taxonomy of the OTU representative sequences, with a confidence threshold set at 0.7 to derive the species taxonomy annotation results.

2.4. Data Analysis

Statistical analysis and data visualization were carried out utilizing SPSS (version 22.0, Chicago, IL, USA), R software (version 4.2.3), Origin 2021 (Origin Lab, Northampton, MA, USA), and Adobe Illustrator CS6. The data were examined through a one-way analysis of variance, accompanied by Duncan’s test for further evaluation (p < 0.05) and a two-way analysis of variance to evaluate the main effects and the interactive effects of the phenolic acid concentration, phenolic acid substance, and processing time. We used redundancy analysis (RDA), Procrustes analysis, correlation analysis, and network analysis to analyze the connection between soil physicochemical properties and microbial communities analyzed using R software (version 4.2.3) with the “vegan” and “corrplot” packages. The Mantel test was calculated with the “linkET” package in R software (version 4.2.3) and the structural equation model (SEM) was developed using AMOS software (version 23.0).

3. Results

3.1. The Impact of Various Treatments on the Chemical Characteristics of the Soil

Soil properties such as available nitrogen (AN), SOM, and pH increased under phenolic acid treatment, while soil AP and AK were decreased with phenolic acid addition (Table 1). The contents of AN, AP, AK, and SOM increased under the high dose of phenolic acid, in comparison to those under the low dose of phenolic acid. The soil AP and SOM were enhanced by 30.08% and 14.16% under the SH_20 treatment, while soil pH decreased by 0.44 units (Table 1). Meanwhile, in comparison to the PL_10 treatment, soil AN and SOM increased by 11.31% and 15.65% under the PH_20 treatment, while soil pH decreased by 0.10 units (Table 1). Moreover, compared to the p-hydroxybenzoic acid treatment, the soil AP and SOM presented different degrees of improvement with salicylic acid addition (Table 1). From the results of multi-way ANOVA, we found the phenolic acid concentration (AN, AK, SOM, and pH), phenolic acid substance (AN, AP, and SOM), and processing time (AP, SOM, and pH) exerted a notable influence on the chemical characteristics of the soil (Table 1).

3.2. Impact of Various Treatments on Soil Extracellular Enzyme Activity

The activities of ACP (acid phosphatase), NAG (1,4-N-acetylglucosaminidase), CBH (cellobiohydrolase), βG (β-1,4-glucosidase), and βX (β-1,4-xylosidase), C/N, C/P, N/P, the vector length, and the vector angle increased with phenolic acid addition (Table 2). Compared to those in the SL_10 treatment, the activities of NAG, ACP, CBH, βG, and βX were significantly increased by 365.38%, 61.22%, 1115.45%, 224.95%, and 965.59% under the SH_20 treatment (Table 2). At the same time, the activities of NAG, CBH, βG, and βX were notably enhanced by 172.72%, 375.03%, 229.69%, and 116.98% under the PH_20 treatment, in comparison to those in the PL_10 treatment (Table 2). In addition, the ratios of C/P and N/P and the vector length and vector angle were significantly increased by 181.52%,189.76%, 17.24%, and 15.72% under the SH_20 treatment, compared to those in the SL_10 treatment (Table 2). Similarly, the ratios of C/P and N/P and the vector length and vector angle increased by 264.80%, 190.48%, 32.00%, and 16.18% (Table 2). Based on the results of the multi-way analysis of variance, the phenolic acid concentration, phenolic acid substance and processing time had a considerable impact on the performance and chemical traits of soil extracellular enzymes (p < 0.05) (Table 2). Moreover, the activities of NAG, CBH, and βX, N/P, and the vector angle were influenced by the interaction effects of the phenolic acid concentration, phenolic acid substance, and processing time (Table 2).

3.3. Effect of Different Treatments Onsoil Microorganisms

We additionally examined how phenolic acid influenced soil micro-organisms. The α diversity of soil microorganisms (bacterium and fungus) decreased with phenolic acid addition (Table S1). Compared to the short-term (10 d) phenolic acid treatment, the ace, chao, and sobs of bacteria significantly decreased with phenolic acid addition under the long-term (20 d) treatment (Table S1). However, the ace, chao and sobs of the bacteria significantly increased under the high-concentration phenolic acid treatment (H), in comparison to the low-concentration phenolic acid treatment (L) (Table S1). It is worth noting that the α diversity of the soil fungi increased with the p-hydroxybenzoic treatment (Table S1).
The main community composition of the bacteria at the phylum level was Firmicutes, Proteobacteria, Actinobacteria, Chloroflexi, and WPS-2 (Figure S1A). Meanwhile, the main community composition of fungi at the phylum level was Ascomycota and Basidiomycota (Figure S1B). Based on the results of PCoA, the soil community composition was separated under different treatments (Figure 1). Furthermore, the processing time of the phenolic acid treatment affected the bacterial community composition, and the concentration of phenolic acid significantly influenced the fungus community composition (Figure 1). LEfse analysis indicated that the type and concentration of phenolic acids could significantly influence the microbial community composition (Figure 2 and Figure S2). Compared to the low dose of salicylic acid, the relative abundance of p_Proteobacteria enhanced and the relative abundance of p_Patescibacteria and p_WPS-2 decreased under a high dose of salicylic acid (Figure S2A,C). Meanwhile, compared to the low dose of p-hydroxybenzoic acid, the relative abundance of p_Proteobacteria increased under the high dose of p-hydroxybenzoic acid treatment, while the relative abundance of p_Chloroflexi, p_Firmicutes, p_Patescibacteria, and p_WPS-2 decreased (Figure S2B,D). In addition, the top five biomarkers of bacteria and fungi were o_Burkholderiales, c_Gammaproteobacteria, p_Proteobacteria, f_Oxalobacteraceae, and g_Massilia with high doses of phenolic acid addition (Figure 2A,B). The top five biomarkers of fungi were g_Trichoderma, f_Hypocreaceae, g_Aspergillus, c_Eurotiomycetes, and o_Eurotiales under the SH_20 treatment (Figure 2C). Additionally, the top five biomarkers of fungi were c_Eurotiomycetes, f_Aspergillaceae, o_Eurotiales, o_Sordariales, and f_Chaetomiaceae under the PH_20 treatment (Figure 2D).

3.4. The Connection Between Soil Characteristics and Microorganisms Across Various Treatments

Based on the results of the RDA, the soil microorganisms were significantly affected by soil properties (Figure 3A,D). The community composition of the bacteria was significantly affected by soil NAG, CBH, βG, βX, AP, SOM, and pH, and the community composition of the fungi was influenced by NAG, ACP, CBH, βG, βX, AP, and pH (Table S2). Additionally, a connection could be observed between soil microorganisms (bacteria, p < 0.05; fungi, p < 0.05) and the soil properties (Figure 3B,E). Furthermore, the relative abundance of Proteobacteria showed a notable positive relationship with soil characteristics (SOM, βG, βX, NAG, and CBH), while the relative abundance of p_Patescibacteria, p_Bdellovibrionota, p_Desulfobacterota, p_Acidobacteriota, p_Chloroflexi, p_Planctomycetota, and p_WPS-2 had a significant negative correlation with soil properties (SOM, βG, βX, NAG, and CBH) (Figure 3C). The relative abundance of p_Mortierellomycota, p_Chytridiomycota, and p_Zoopagomycota had a significant negative relationship with SOM, βG, βX, NAG, and CBH (Figure 3E). The α-diversity of bacteria had a positive correlation with NAG and βG, while the α-diversity of fungi had a positive correlation with NAG, ACP, CBH, βX, AP, and SOM with salicylic acid addition (Figure 4A). Moreover, the α-diversity of bacteria presented a positive correlation with ACP, βX, and AK, and the α-diversity of fungi had a positive correlation with βG, AN, and SOM with p-hydroxybenzoic acid addition (Figure 4B).
The type, concentration, and processing time of phenolic acid exerted a considerable influence on soil chemical characteristics and microbial diversity, as indicated by the structural equation model (SEM) (Figure 5). The concentration and processing time of phenolic acid presented a notable positive correlation (p < 0.05) with soil extracellular enzymes (0.823, time; concentration,0.347) and soil chemical properties (0.347, time; concentration,0.612) (Figure 5A). However, the type of phenolic acid had a negative influence (p < 0.05) on soil extracellular enzymes (−0.624) (Figure 5A). In addition, the processing time of phenolic acid showed a strong positive correlation with fungal diversity and the concentration of phenolic acid exhibited a notably positive association with bacterial diversity (Figure 5A). Based on the results of total effect, we found that the influencing factors (the processing time of phenolic acid and soil chemical properties) had a significant impact on bacterial diversity, and the soil extracellular enzymes had a significant impact on fungus diversity (Figure 5B,C). The findings from the two-factor network analysis revealed a strong relationship between soil microorganisms and the chemical properties of the soil (Figure 6). The p_Verrucomicrobiota, p_Patescibacteria, p_WPS2, and p_Mortierellomycota were important soil microorganisms with salicylic acid addition (Figure 6A,B). The relative abundance of p_Verrucomicrobiota demonstrated a positive correlation with soil NAG, ACP, CBH, βG, βG, and AP, while the relative abundance of p_Patescibacteria and p_WPS2 had a negative relationship with soil chemical properties (Figure 6A). The p_Gemmatimonadota, p_Patescibacteria, p_Actinobacteriota, p_Proteobacteria, p_Firmicutes, and p_Mortierellomycota were important soil microorganisms with p-hydroxybenzoic acid addition (Figure 6C,D). Soil bacteria exhibited a clear positive correlation with soil chemical properties, while soil fungi exhibited a marked negative relationship with these characteristics (Figure 6C,D).

4. Discussion

4.1. Influence of Various Treatments on Chemical Characteristics of Soil

This study revealed that phenolic acid type, concentration, and treatment time had varying influences on the soil’s physicochemical characteristics. Compared to the control, phenolic acid application reduced the soil AN and AP contents (Table 1). This result may be explained by the fact that autotoxic substances present in root exudates contribute to the deterioration of soil physical properties (soil compaction, increased bulk density, reduced ventilation, and water permeability), which decreases soil nutrient availability [25]. In addition, excessive soil acidity can hinder enzyme activity, thereby diminishing the availability of essential nutrients in the soil [26]. The soil pH significantly decreased with a high dose of phenolic acid (Table 1). A possible explanation for this might be that salicylic acid and benzoic acid are prevalent root exudates, and their accumulation can contribute to soil acidification [27]. Earlier research has shown that the buildup of phenolic acids in soil subjected to cropping contributes to soil acidification [28]. In addition, adding moderate amounts of phenolic acids improves nutrient availability and organic matter in soil (Table 1). The organic acid anion replaces the phosphate anion, forming a complex with the metal ion, which enhances the solubility of insoluble nutrients, thereby improving nutrient availability [29]. In the rhizosphere zone, hydrogen ions in the soil dissolve the original crystal lattice that has been disrupted by weathering and cracking, effectively replacing the potassium within it and enhancing the availability of potassium in the soil [30]. Guava acid and citric acid present in leguminous root exudates enhance the availability of insoluble phosphorus [31].
In comparison to the absence of phenolic acid, the application of phenolic acid resulted in a significant increase in soil extracellular enzyme activity (Table 2). It is possible that root exudates can supply specific carbon sources and energy substrates for the growth and reproduction of microorganisms, while also stimulating the release of extracellular enzymes [32]. Additionally, root exudates can also function as ligands, directly activating or dissolving protected organic matter, which makes it accessible for microbial utilization and enhancing enzyme activity [33]. In addition, high concentrations of phenolic acids can also enhance soil extracellular enzyme activity, especially in CBH, βG, and βX, compared to low concentrations of phenolic acids (Table 2). These findings suggest that the addition of phenolic acids can stimulate the growth of specific microbial groups and enhance the release of extracellular enzymes associated with carbon conversion [34].

4.2. Effect of Different Treatments on Soil Microorganisms

Compared to the control, the addition of phenolic acid significantly reduced soil microbial diversity (Table S1). In the plant rhizosphere, microorganisms utilize amino acids, fatty acids, organic acids, and other substances released by the roots as nutritional carbon and nitrogen sources, enabling them to colonize the rhizosphere in substantial numbers. Additionally, the concentration of phenolic acids can significantly influence microbial diversity and community composition (Table S1; Figure 1). Kamilova et al. [35] found that bacterial communities capable of utilizing root exudates significantly differ from other microbial communities. Phenolic acids exert a concentration-dependent effect on soil microbial diversity and plant growth, demonstrating a pattern of low promotion at lower concentrations and significant suppression at higher concentrations [17]. Benzoic acid (≤2 μg/L) and phenylpropionic acid (≤3 μg/L) can stimulate the growth of R. solanacearum and Brevibacillus brevis at low concentrations, while the growth of microorganisms is inhibited with the concentration of these acids increases [18]. Salicylic acid p-hydroxybenzoic acid and can enhance plant growth (with a low concentration of 0.01 g/mL), while plant growth is inhibited with a high concentration of phenolic acids [36].
Different phenolic acid substances had different effects on microbial diversity (Table S1; Figure 1). A possible explanation for this result that different phenolic acid compounds possess varying chemical functional groups and distinct energy properties, which leads to significant differences in the capacity of soil microbial groups to utilize the various phenolic acid compounds [37,38]. For example, oxalic acid addition increases Gram-negative bacteria and the total microbial phospholipid fatty acid content, whereas the addition of glycine significantly decreases the abundance of Gram-positive bacteria, actinomycetes, and the activity of the microbial community [39]. This is primarily because glycine is classified as an amino acid, and only a select few microbial groups are capable of utilizing glycine [40,41]. In our study, the relative abundance of p_Proteobacteria increased, while the relative abundance of p_Patescibacteria and p_WPS-2 decreased under high concentrations of salicylic acid compared to low concentrations (Figure S2). p_Patescibacteria and p_WPS-2 are prevalent beneficial microorganisms in the soil that engage in various essential biological processes, significantly contributing to nutrient cycling and metabolism [42,43]. Phenolic acids may act as chemotactic signals, enhancing motility and pathogenicity in Proteus, which could explain the dominance of Proteobacteria under high phenolic acid concentrations [44]. In addition, phenolic acids addition enhanced the availability of phosphorus in the soil in this study, potentially due to the enrichment of o_Burkholderiales and g_Massilia within the soil (Table 1 and Figure 2). Marseille and Burkholderia are significant beneficial bacteria that play crucial roles in nitrogen fixation, nodulation, phosphorus solubilization, and the production of plant hormones [45]. In addition, the relative abundances of g_Aspergillus, c_Eurotiomycetes, and o_Eurotiales were enriched with high doses of phenolic acids (Figure 2C,D). Aspergillus serves a phosphorus-dissolving function by converting insoluble forms of phosphorus in soil into soluble phosphorus and enhancing the availability of phosphorus for absorption by plant roots [46]. Thus, as a crucial medium for information exchange, plant root exudates facilitate effective interactions between microorganisms and plants, while also promoting the co-evolution of individual microorganisms and their host plants.

4.3. The Relationship Between Soil Properties and Microbial Communities Under Various Treatment

Previous studies have demonstrated a significant correlation among phenolic acids and soil’s physical and chemical attributes, along with its microbial communities [12,47]. In this study, phenolic acids could significantly influence the composition of microbial communities by indirectly affecting the physical properties of the soil (Table S2 and Figure 3). Soil acidification resulted in a decline in the population of microorganisms, as well as reductions in microbial respiration, activity, and metabolic entropy, which can subsequently impact the nutrient recycling processes in which soil microorganisms are involved [48]. In addition, soil acidification can result in a decline in soil fertility, impact the availability of soil nutrients and enzyme activity, and indirectly influence the diversity of soil microorganisms [49,50]. In comparison to bacteria diversity, fungi diversity exhibited a closer relationship with the chemical properties of soil following the addition of salicylic acid. (Figure 4A). A possible explanation for this might be that bacterial soil serves as a biological indicator of improved soil fertility, while fungal soil signifies the depletion of soil fertility [51]. Li et al. [52] demonstrated that the long-term continuous cropping of peanuts can alter the composition of soil biological communities, resulting in an increase in pathogenic fungi. In addition, the processing time and concentration of phenolic acid showed a strong positive correlation with the diversity of soil microorganisms (Figure 5). The allelochemicals continuously secreted by root systems do not directly cause toxic effects; rather, their impact is mediated through alterations in the soil microbial [53]. With the gradual accumulation of peanut root exudates, the population of peanut pathogenic fungus was significantly increased in the soil [54]. Generally, low concentrations of root exudates promote physiological and biochemical metabolism as well as plant growth, while high concentrations may inhibit these processes [55]. In addition, phenolic acid selectively enriched p_Patescibacteria, and p_Mortierellomycota (Figure 6). p_Mortierellomycota is classified as a saprophytic fungus and serves as a beneficial soil microorganism that is essential for the transformation of carbon and nutrients in the soil [56]. The relative abundance of p_Patescibacteria also experienced a significant change in the continuous cropping obstacle soil of Andrographis paniculata [57].

5. Conclusions

This study utilized high-throughput sequencing to explore how soil physical and chemical characteristics interact with the microbial environment under the influence of phenolic acid treatments. The findings reveal that phenolic acids gradually lowered soil pH through cumulative mechanisms, thereby influencing the availability of soil nutrients. Compared to the control, the extracellular enzyme activity in the soil exhibited varying degrees of enhancement with phenolic acid addition. However, the application of phenolic acid reduced soil microbial diversity. Moreover, in comparison to low-concentration phenolics, treatment with high-concentration phenolics could enhance the diversity of soil microorganisms. Through principal component analysis, correlation analysis, and structural equation modeling, it was found that there was a strong correlation between soil chemical properties and microorganisms. Additionally, the concentration of phenolic acids exhibited a significant positive correlation with microbial diversity. Phenolic acids influence the soil ecological environment by altering the relative abundance of functional microorganisms (p_Patescibacteria and p_Mortierellomycota) in the soil. Reasonable rotation, intercropping, and the application of appropriate soil amendments can mitigate phenolic acid accumulation, thereby restoring soil microecological balance and enhancing agricultural sustainability.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture15101067/s1, Figure S1: The composition of the microbiome communities of the different groups at the phylum; Figure S2: The LEfSe analysis of soil microbiome communities under different treatments (LDA > 4). (A and C: SL_10 vs. SH_20; B and D: PL_10 vs. SH_20); Table S1: Effect of different treatments on the α diversity of soil microorganism; Table S2: The environment factors in RDA analysis between soil microorganisms and chemical property.

Author Contributions

H.X.: data curation, supervision, funding acquisition, and writing—original draft; M.R. and Z.E.-D.: review and editing; Y.L., C.W., X.Z., J.D., Z.C. and H.L.: supervision; J.S. and C.J.: writing—original draft; Y.Z.: writing—original draft, review and editing, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the Anhui Academy of Agricultural Sciences Academic Research Plan Project (grant number 2025YL030); the Anhui Province College Students Innovative Entrepreneurial Training Plan Program (grant number S202410879287); and the Key Project of Education Department of Anhui Province (grant number 2024AH050295).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

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

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The PCoA of soil microbial communities using the Bray–Curtis distances under different treatments ((A,C) salicylic acid; (B,D) p-hydroxybenzoic acid).
Figure 1. The PCoA of soil microbial communities using the Bray–Curtis distances under different treatments ((A,C) salicylic acid; (B,D) p-hydroxybenzoic acid).
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Figure 2. The LDA analysis of soil microbial communities between different phenolic acid treatments (LDA > 4). ((A,C) SL_10 vs. SH_20; (B,D) PL_10 vs. SH_20.).
Figure 2. The LDA analysis of soil microbial communities between different phenolic acid treatments (LDA > 4). ((A,C) SL_10 vs. SH_20; (B,D) PL_10 vs. SH_20.).
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Figure 3. The redundancy analysis (RDA), Procrustes analysis, and correlation analysis of soil microorganisms and soil metabolites under different varieties. p < 0.05 (*); p < 0.01 (**); and p < 0.001 (***).
Figure 3. The redundancy analysis (RDA), Procrustes analysis, and correlation analysis of soil microorganisms and soil metabolites under different varieties. p < 0.05 (*); p < 0.01 (**); and p < 0.001 (***).
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Figure 4. The Mantel test was used to examine the relationships among various environmental factors across different treatments. The heat map illustrates the Pearson correlations among these factors. Line thickness denotes the magnitude of the Mantel correlation coefficient, while line color reflects the corresponding p-value. Significance levels are as follows: * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 4. The Mantel test was used to examine the relationships among various environmental factors across different treatments. The heat map illustrates the Pearson correlations among these factors. Line thickness denotes the magnitude of the Mantel correlation coefficient, while line color reflects the corresponding p-value. Significance levels are as follows: * p < 0.05; ** p < 0.01; *** p < 0.001.
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Figure 5. SEMs illustrate both the direct and indirect effects of each type, concentration, and processing time of phenolic acid, soil extracellular enzymes, and soil chemical properties on the bacterial and fungus diversity (A). The causal links, both direct and indirect, between the influencing variables and microbial diversity (including soil bacteria (B) and fungi (C)) are illustrated. The black solid lines represent standardized coefficients corresponding to each path in the model. * p < 0.05 and *** p < 0.001.
Figure 5. SEMs illustrate both the direct and indirect effects of each type, concentration, and processing time of phenolic acid, soil extracellular enzymes, and soil chemical properties on the bacterial and fungus diversity (A). The causal links, both direct and indirect, between the influencing variables and microbial diversity (including soil bacteria (B) and fungi (C)) are illustrated. The black solid lines represent standardized coefficients corresponding to each path in the model. * p < 0.05 and *** p < 0.001.
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Figure 6. Network visualization of the relationship between soil microorganisms (soil bacteria (A,C) and fungi (B,D)) and physicochemical properties under different phenolic acid treatments. Node size reflects the centrality score, where genera with higher centrality are identified as key species within the network. Connections between nodes signify strong interactions—positive (shown in red) or negative (shown in green).
Figure 6. Network visualization of the relationship between soil microorganisms (soil bacteria (A,C) and fungi (B,D)) and physicochemical properties under different phenolic acid treatments. Node size reflects the centrality score, where genera with higher centrality are identified as key species within the network. Connections between nodes signify strong interactions—positive (shown in red) or negative (shown in green).
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Table 1. The impact of various treatments on the chemical characteristics of the soil.
Table 1. The impact of various treatments on the chemical characteristics of the soil.
TreatmentAN (mg/kg)AP (mg/kg)AK (mg/kg)SOM (%)pH
CK51.36 ± 1.20 e63.04 ± 1.38 a180.06 ± 1.91 a1.08 ± 0.02 g6.48 ± 0.12 b
SL−1062.11 ± 0.91 d30.19 ± 1.84 d169.11 ± 1.88 c1.13 ± 0.04 fg6.60 ± 0.13 a
SM−1062.73 ± 2.14 cd30.83 ± 4.00 d170.21 ± 3.76 bc1.20 ± 0.01 cde6.36 ± 0.07 ab
SH−1061.82 ± 0.67 d31.37 ± 2.09 d172.53 ± 4.84 bc1.21 ± 0.02 cd6.23 ± 0.09 cd
SL−2062.61 ± 2.75 cd31.51 ± 5.71 d171.37 ± 3.09 bc1.19 ± 0.02 cde6.43 ± 0.09 ab
SM−2062.58 ± 1.24 cd34.92 ± 4.03 cd173.53 ± 2.05 bc1.23 ± 0.05 c6.38 ± 0.02 ab
SH−2062.64 ± 1.94 cd39.27 ± 5.31 c174.60 ± 3.43 abc1.29 ± 0.02 ab6.16 ± 0.02 d
PL−1063.39 ± 0.59 cd45.94 ± 1.71 b170.21 ± 4.97 bc1.15 ± 0.02 ef6.41 ± 0.02 ab
PM−1065.66 ± 1.63 bc46.96 ± 2.23 b171.48 ± 5.52 bc1.17 ± 0.02 def6.38 ± 0.05 ab
PH−1069.54 ± 2.50 a47.25 ± 1.08 b172.84 ± 1.79 bc1.23 ± 0.02 bc6.35 ± 0.04 bc
PL−2065.69 ± 1.42 bc46.57 ± 2.25 b172.81 ± 1.74 bc1.29 ± 0.02 ab6.36 ± 0.01 ab
PM−2066.55 ± 0.78 b47.75 ± 1.18 b175.65 ± 1.90 abc1.31 ± 0.06 a6.33 ± 0.04 bc
PH−2070.56 ± 1.73 a48.22 ± 3.46 b176.77 ± 5.03 ab1.33 ± 0.04 a6.31 ± 0.02 bc
Phenolic acid types64.67 **167.56 **0.3712.15 **0.08
Concentration8.03 **2.508.87 **16.86 **28.48 **
Time2.585.77 **0.3877.78 **7.86 **
P × C9.37 **0.662.871.1914.23 **
P × T0.842.791.6010.55 **0.44
C × T0.290.853.54 **0.141.99
P × C × T0.170.693.141.471.98
Note: Significant differences across different years of continuous cropping are indicated by lowercase letters, based on Duncan’s test (p < 0.05). The results are based on three replicates (n = 39); p < 0.01 is marked as **.
Table 2. Impact of various treatments on soil extracellular enzyme activity.
Table 2. Impact of various treatments on soil extracellular enzyme activity.
TreatmentNPCChemometric Characteristics
NAGACPCBHβGβXC/NC/PN/PVector LengthVector Angle
CK50.08 ± 1.31 g57.94 ± 2.62 e10.19 ± 0.60 d30.20 ± 1.18 f2.63 ± 1.03 d0.86 ± 0.03 e0.74 ± 0.05 g0.87 ± 0.03 d0.63 ± 0.02 e42.68 ± 0.66 g
SL−1088.51 ± 1.89 f69.47 ± 1.10 d12.36 ± 1.05 d107.72 ± 14.65 e7.47 ± 1.06 cd1.44 ± 0.21 c1.84 ± 0.26 e1.27 ± 0.01 c0.87 ± 0.05 c47.65 ± 0.29 de
SH−1096.56 ± 1.91 ef84.08 ± 2.23 c13.33 ± 1.49 d190.60 ± 24.51 d9.95 ± 2.48 cd2.22 ± 0.25 a2.54 ± 0.26 d1.15 ± 0.05 cd0.99 ± 0.03 a46.19 ± 0.44 f
SL−20202.58 ± 1.20 c94.82 ± 17.4840.71 ± 11.23 c301.90 ± 9.17 b19.63 ± 4.21 bc1.77 ± 0.06 b4.15 ± 0.38 b2.35 ± 0.26 b1.03 ± 0.01 a51.57 ± 0.70 c
SH−20411.91 ± 28.75 a112.00 ± 1.10 a150.23 ± 3.31 a350.04 ± 31.28 a79.60 ± 19.73 a1.42 ± 0.22 c5.18 ± 0.44 a3.68 ± 0.29 a1.02 ± 0.03 a55.14 ± 1.46 b
PL−10107.43 ± 1.76 de85.14 ± 1.11 c16.94 ± 2.11 d79.81 ± 5.25 e9.60 ± 2.81 cd0.99 ± 0.06 de1.25 ± 0.07 f1.26 ± 0.01 c0.75 ± 0.02 d48.15 ± 0.19 d
PH−10117.50 ± 5.90 d98.45 ± 4.56 b26.01 ± 4.95 cd177.78 ± 11.42 d8.97 ± 1.42 cd1.81 ± 0.16 b2.16 ± 0.04 de1.20 ± 0.09 cd0.94 ± 0.02 b46.72 ± 0.80 ef
PL−20215.24 ± 5.15 c63.24 ± 5.83 de36.83 ± 1.76 c162.23 ± 12.75 d23.15 ± 2.35 b1.03 ± 0.09 de3.52 ± 0.15 c3.43 ± 0.38 a0.93 ± 0.01 b56.90 ± 1.23 a
PH−20292.98 ± 4.33 b80.02 ± 2.01 c80.47 ± 27.30 b263.13 ± 35.01 c20.83 ± 4.29 bc1.24 ± 0.05 cd4.56 ± 0.22 b3.66 ± 0.15 a0.99 ± 0.01 a55.94 ± 0.35 ab
Phenolic acid types14.40 **13.25 **10.39 **61.73 **19.60 **47.42 **27.15 **11.02 **58.48 **29.64 **
Concentration304.33 **98.79 **87.07 **99.28 **23.70 **32.29 **74.64 **17.38 **80.42 **0.05
Time1659.67 **0.51187.67 **238.35 **76.94 **15.25 **510.02 **622.56 **103.82 **550.08 **
P × C54.86 **1.8810.91 **3.6328.63 **5.74 *0.269.83 **11.91 **11.72 **
P × T69.78 **148.73 **27.01 **29.44 **21.29 **0.040.419.68 **1.7215.06 **
C × T236.34 **3.9666.96 **0.6920.84 **45.94 **1.1128.34 **40.31 **17.54 **
P × C × T58.34 **1.0117.89 **1.0123.45 **3.940.2212.18 **0.0412.01 **
Note: Significant differences across different years of continuous cropping are indicated by lowercase letters, based on Duncan’s test (p < 0.05). The results are based on three replicates (n = 39); p < 0.05 and p < 0.01 are marked as * and **.
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Zhou, Y.; Liu, Y.; Jiang, C.; El-Desouki, Z.; Riaz, M.; Wang, C.; Zhang, X.; Ding, J.; Chen, Z.; Liu, H.; et al. Effects of Exogenous Application of Phenolic Acid on Soil Nutrient Availability, Enzyme Activities, and Microbial Communities. Agriculture 2025, 15, 1067. https://doi.org/10.3390/agriculture15101067

AMA Style

Zhou Y, Liu Y, Jiang C, El-Desouki Z, Riaz M, Wang C, Zhang X, Ding J, Chen Z, Liu H, et al. Effects of Exogenous Application of Phenolic Acid on Soil Nutrient Availability, Enzyme Activities, and Microbial Communities. Agriculture. 2025; 15(10):1067. https://doi.org/10.3390/agriculture15101067

Chicago/Turabian Style

Zhou, Yi, Yihang Liu, Chaoqiang Jiang, Zeinab El-Desouki, Muhammad Riaz, Chenlu Wang, Xueping Zhang, Jiayi Ding, Zhenghao Chen, Huaiwei Liu, and et al. 2025. "Effects of Exogenous Application of Phenolic Acid on Soil Nutrient Availability, Enzyme Activities, and Microbial Communities" Agriculture 15, no. 10: 1067. https://doi.org/10.3390/agriculture15101067

APA Style

Zhou, Y., Liu, Y., Jiang, C., El-Desouki, Z., Riaz, M., Wang, C., Zhang, X., Ding, J., Chen, Z., Liu, H., Shen, J., & Xia, H. (2025). Effects of Exogenous Application of Phenolic Acid on Soil Nutrient Availability, Enzyme Activities, and Microbial Communities. Agriculture, 15(10), 1067. https://doi.org/10.3390/agriculture15101067

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