Next Article in Journal
Advancements on the Mechanism of Soluble Sugar Metabolism in Fruits
Previous Article in Journal
Clonal Micropropagation of Promising Genotypes of Amygdalus communis L. for Population Restoration and Gene Pool Conservation
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Horticulturae
Volume 11
Issue 9
10.3390/horticulturae11091000
horticulturae-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

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

Strawberry Performance and Rhizospheric Health Were Efficiently Improved After Long-Term Sheep Manure Organic Fertilizer Application

by
Zhengyan Chou
1,2,
Chenghao Lei
1,
Xinyi Cai
1,
Yong Li
3,
Diya Zeng
1,2,
Sidan Gong
3,
Jianping Wang
3 and
Zhilian Gong
1,2,*
1
College of Food and Biological Engineering, Xihua University, Chengdu 610039, China
2
Food Microbiology Key Laboratory of Sichuan Province, Xihua University, Chengdu 610039, China
3
College of Environmental Science and Engineering, Southwest Jiaotong University, Chengdu 610059, China
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(9), 1000; https://doi.org/10.3390/horticulturae11091000 (registering DOI)
Submission received: 15 July 2025 / Revised: 13 August 2025 / Accepted: 19 August 2025 / Published: 23 August 2025
(This article belongs to the Section Plant Nutrition)
Browse Figures
Review Reports Versions Notes

Abstract

Strawberry is a popular fruit with great commercial value. It is meaningful to study how to improve strawberry yield and quality in a sustainable way. In this research, the potential impacts of replacing chemical fertilizer (CF) with sheep manure organic fertilizer (SMOF) on strawberry rhizospheric bacteria, soil physicochemical properties, strawberry fruit yield, and nutritional quality were studied through a strawberry field experiment with 16 years of different fertilizer applications. This study showed that, compared with chemical fertilizer, SMOF effectively improved soil physicochemical properties and increased the relative abundance of beneficial bacteria, the absolute abundance of phosphorus-related functional genes pqqC and phoD and bacteria diversity, and enhanced synergistic action among strawberry rhizospheric bacteria. The yield, and the contents of total soluble solids, soluble sugar, soluble protein, and vitamin C, and sugar/acid ratio of strawberry fruit in SMOF treatment were significantly higher than in CF treatment by 40%, 21%, 15%, 46%, 23%, and 41%, respectively (p < 0.05). Pearson correlation coefficient analysis showed that strawberry fruit yield and nutritional quality were positive with soil pH, bacterial diversity, soil enzyme activity, and nutrient content, and negative with soil density. The results showed that long-term SMOF could efficiently improve strawberry performance and rhizospheric health.

1. Introduction

In order to improve crop yield, a large quantity of chemical fertilizers was applied. Excessive application of chemical fertilizer (CF) was not only expensive in agricultural production, but also led to such challenges as soil compaction, acidification, soil microbial diversity decrease, and poor quality of agricultural products [1,2]. In contrast, organic fertilizer application, especially manure organic fertilizer, could alleviate manure harm to the environment and the dependence of agricultural production on CF, which reaped good environmental and economic benefits [3,4]. Furthermore, manure organic fertilizer, rich in organic matter and a variety of nutrients, could effectively improve soil pH, and water and fertilizer conditions [3,4]. Compared with CF, organic fertilizer was more effective in promoting crop growth and increasing nutritional quality of agricultural products by improving soil physicochemical properties [5,6]. Previous studies also found that manure organic fertilizer application was beneficial to improving soil microbial community structure and microbial activity, thus increasing crop yield and quality [7,8]. Therefore, the application of organic fertilizer, especially manure fertilizer application in agricultural production, had more and more importance attached to it.
Strawberry was a very popular and commercially important fruit, with a planting area of up to 389,665 ha and 9,175,384 tons in the world. Of all countries, China ranked first, with the production of 3,380,478 tons [9]. It is of great importance to explore how to increase strawberry yield and nutritional quality in a sustainable way.
With the improvement of people’s living standard and environmental awareness, although CF application to obtain high strawberry yield was still popular, more and more studies focused on organic fertilizer application onto strawberry planting. Nakielska et al. (2024) [10] found that microbial organic fertilizer could effectively improve the yield and economic effectiveness of organic strawberry production, with the profitability index of 129–169%. Vishwakarma et al. (2024) [11] found that application of chemical fertilizer with the aid of manure organic and bio-organic fertilizer could help farmers to produce quality strawberry fruits and higher profitability. However, the effectiveness of microbial organic fertilizer application was limited to plantation conditions [10]. Previous studies also found that sheep manure organic fertilizer (SMOF) was a more effective strategy than bio-organic fertilizer (algae-optimized bacteria) to promote strawberry growth by ameliorating soil physicochemical conditions and microbe community structure [12]. Combined application of poultry manure organic fertilizer and CF could significantly increase strawberry yield, fruit quality, and economic viability by improving soil pH, soil nutrient content, and strawberry nutrient uptake [13]. However, most of the studies focused on short-term organic fertilizer application to strawberry planting.
At present, the impacts of long-term replacement of CF with livestock manure organic fertilizer on strawberry rhizospheric health, yield, and quality still remain unclear. In this study, strawberry field experiment sites with 16 year’s of different fertilization application were selected to study the impacts of long-term replacement of CF with SMOF on strawberry rhizospheric bacteria, soil physicochemical properties, strawberry yield, and nutrition quality. We hypothesized that long-term SMOF application to strawberries could efficiently achieve the following: (1) improve soil physicochemical properties; (2) enhance strawberry rhizospheric bacteria diversity and structure improvement; and (3) increase strawberry yield and nutritional quality.

2. Materials and Methods

2.1. Study Site Description

Two strawberry field experiment bases with different fertilizer applications were chosen in Wangsi Town, Dayi County, Sichuan Province, China (103°28′ E, 30°31′ N, 548 m a.s.l.). The study site was located in a subtropical humid climate area, with annual average precipitation of 1000 mm and annual average temperature of 16.1 °C. The study was initiated in 2009. Composite inorganic chemical fertilizer (N:P2O5:K2O = 15:15:15), purchased from Guizhou Xiyang Industrial Co., Ltd. (Guizhou, China), was applied in one base with the amount of 300 kg/hm2. In the other base, SMOF was applicatied with the amount of 2500 kg/hm2. SMOF produced by fermenting sheep manure naturally was purchased from Hongyuan County, Aba Prefecture, Sichuan Province. Nutrient contents of SMOF were as follows: organic matter 31.82%, N 1.76%, P2O5 1.68%, and K2O 1.85%. Two strawberry bases were 1.0–1.5 km apart. Strawberry variety was Hongyan. Except for different fertilizer application, the other field management measures were the same in two bases.

2.2. Sample Collection

In 2024–2025 season, three 10 m × 10 m plots were randomly set up in each strawberry base, with the distance of no less than 50 m among them. In each plot, 5 strawberry plants were randomly chosen for composite sample collection of strawberry leaves and rhizospheric soil. In December 2024, when the first generation strawberry fruit matured, about 500 g composite rhizospheric soil was sampled by shaking off method and brought back to the laboratory with an ice bag. Some were stored at −80 °C for 16S rDNA high-throughput sequencing of soil micro-organisms and quantitative PCR (qPCR) analysis of phosphorus (P)-related functional genes (pqqC and phoD). The other soil samples were air-dried, ground, and passed through a soil screen with a pore size of 2 mm for determination of soil physicochemical properties and soil enzyme activities. In each plot, in order to sample representative fruit samples, about 400 g ripe strawberry fruits were collected randomly as a composite fruit sample. Samples of fresh leaves and strawberry fruits were brought back to the laboratory with an ice bag and stored at −20 °C for determining the contents of photosynthetic pigments and malondialdehyde (MDA), and antioxidant enzyme activities and strawberry fruit nutritional quality. Five strawberry plants were randomly chosen from each sample plot for determining strawberry fruit yield by picking and weighing ripe strawberry fruit during 2024–2025 harvest season.

2.3. Strawberry Rhizospheric Soil pH, Bulk Density, Soil Nutrient Content, and Soil Enzyme Activity

Soil pH was measured using a pH meter (soil:water = 1:2.5) (Thunder magnetic PHSJ-3F, Shanghai Yidian Scientific Instruments Co., Ltd., Shanghai, China). Soil bulk density was determined by ring sampler [14]. Soil nutrient contents were determined according to the description of Lu (2000) [15]. The contents of soil TN, soil alkali-hydrolyzable nitrogen (AN), soil organic carbon (SOC), and soil available phosphorus (AP) were determined by Kjeldahl method (K1100, Haineng Future Technology Group Co., Ltd., Jinan, China), alkali-hydrolysis diffusion method, potassium dichromate oxidation method, and molybdenum antimony colorimetry (TU-1810, Beijing Puxi General Instruments Co., Ltd., Beijing, China), respectively [15]. Soil enzyme activities were determined according to the description of Guan (1986) [16]. Soil urease (Ure), alkaline phosphatase (ALP), and sucrase (Suc) activities were determined by sodium phenol-sodium hypochlorite colorimetry, disodium phenyl phosphate colorimetry, and 3,5-dinitrosalicylic acid colorimetry [16].

2.4. Bacterial Community Structure and qPCR Analysis of P-Related Genes (pqqC and phoD) in Strawberry Rhizospheric Soil

The bacterial community structure of strawberry rhizospheric soil was investigated by 16S rDNA high-throughput sequencing. The primers for PCR amplification were 338F (5-ACCCTACGGGGGCAG-3′) and 806R (5′-GGACTACHVGGTWTCTAAT-3′). According to the method of Li et al. [17], the absolute abundance of P-related functional genes (pqqC and phoD) was determined by qPCR technology. The primers ApqCF (5-AACCGCTTACTCAG-3) and ARpqCR (5-GCGAACAGCTCGGTCAG-3) were used for pqqC gene and the primers ALPS-F730 (5-CAGTGGGACGCACGGT-3) and ALPS-1101 (5-GAGGCCGATCGGCATGTCG-3) were for phoD gene. High-throughput 16S rDNA high-throughput sequencing and qPCR of P-related functional genes were completed by Majorbio Bioinformatics Technology Co., Ltd. (Shanghai, China).

2.5. Determination of Antioxidant Enzyme Activity, MDA, and Photosynthetic Pigment Content in Strawberry Leaves

Antioxidant enzyme activity of strawberry leaves was determined according to the description of Li (2000) [18]. Superoxide dismutase (SOD) activity was determined by nitrogen blue tetrazole method and 50% inhibition of NBT photochemical reduction was defined as an enzyme activity unit. Catalase (CAT) activity was determined by ultraviolet absorption method and 0.1 decrease in OD240 in 1 min was defined as an enzyme activity unit. Peroxidase (POD) activity was determined by guaiacol oxidation method [18], and the change of OD470 in 1 min was 0.01 as an enzyme activity unit. MDA content was determined by thiobarbituric acid method [18]. Chlorophyll a (Chl a), Chlorophyll b (Chl b), and carotenoid (Car) were determined by ultraviolet spectrophotometry (TU-1810, Beijing Puxi General Instruments Co., Ltd., Beijing, China) [18].

2.6. Determination of Plant Biomass and Fruit Nutritional Quality of Strawberry

The biomass of strawberry plants was determined by drying method. The contents of soluble sugar, soluble protein, and vitamin C in strawberry fruits were determined by anthrone-ethyl acetate colorimetry, Coomassie brilliant blue G-250 staining method, and 2,6-dichloroindophenol oxidation titration method, respectively [18]. Titratable acid content was determined by acid-base titration. Total soluble solids (TSS) content is measured by handheld refractometer (PR-32α, ATAGO, Tokyo, Japan). Sugar–acid ratio was the ratio of soluble sugar content and titratable acid content.

2.7. Data Analysis

In order to explore the effect of different fertilizer applications on soil rhizospheric health, strawberry growth, strawberry fruit yield, and quality, independent sample t-test analysis was performed with SPSS 23.0 (IBM SPSS Inc., Chicago, IL, USA). The links of soil nutrients with soil pH, soil bulk density, soil enzyme activity, soil bacterial composition, and abundance of P-related genes were analyzed by Pearson correlation coefficient analysis with Origin 2024. The links of strawberry fruit yield and quality with soil physicochemical properties, soil bacterial diversity, and strawberry leaf physiological indices were also analyzed by Pearson correlation coefficient analysis. There were 3 replicates in each treatment, and the significance level of the difference was set at p = 0.05.

3. Results and Discussion

3.1. Effects of Long-Term SMOF on Microbial Community Structure and P-Involved Functional Gene Abundance in Strawberry Rhizospheric Soil

Soil microbes are an important component of soil ecosystems, and their community composition and diversity were the key indicators for evaluating the ecological function of soil microbial community [19]. The results of 16S rDNA high-throughput sequencing showed that the dominant bacteria phyla in strawberry rhizospheric soil mainly included Proteobacteria, Firmicutes, Chloroflexi, Acidobacteriota, Actinobacteriota, and Bacteroidota, accounting for about 85–88% of the total abundance (Figure 1a). At genus level, the dominant genera in both treatments mainly included Bacillus, Sphingomonas, and norank genera of Gemmatimonadetes (Figure 1b). In addition, norank genera of Acidobacteriota were the dominant genera in CF treatment, which might be related to soil acidification caused by chemical fertilizer application. Norank genera of Chloroflexi were the dominant genera in SMOF treatment, which might be attributed to rich organic matter in SMOF.
It was worth noting that SMOF significantly increased the relative abundance of beneficial bacteria in strawberry rhizospheric soil. At phyla level, compared with CF treatment, SMOF treatment increased the relative abundance of Proteobacteria, Chloroflexi, and Bacteroidota by 40%, 24% and 103%, respectively. At genus level, SMOF efficiently increased Sphingomonas genus and several norank genera of Chloroflexi. Proteobacteria had significant morphological and physiological diversity and played an important role in nutrient cycles of soil nitrogen, P, and potassium [20]. Most members of Proteobacteria and Bacteroidota had been proved with plant growth-promoting abilities [17]. They contained genes encoded with phosphatases or gluconic acid synthesis, which mineralized organic phosphorus or dissolved phosphate and increased soil AP content [21,22]. Chloroflexi was a group of micro-organisms rich in nutrient metabolic pathways, involved in nutrient cycles such as carbon and nitrogen [23]. Sphingomonas was reported to have plant growth-promoting properties such as being involved in the nitrogen cycle [24].
Notably, it was found for the first time that long-term SMOF significantly increased the absolute abundance of phoD and pqqC genes in strawberry rhizospheric soil (Figure 1c). The gene phoD encoded alkaline phosphatase, which was involved in mineralizing soil organic phosphorus [25]. Bacteria containing phoD gene secreted ALP to catalyze organic phosphorus mineralization and promoted P release [26,27]. Pyrroloquinoline quinone (pqq) is an important cofactor in gluconic acid synthesis involved in dissolving inorganic phosphate [28]. The gene pqqC was an important gene encoding pqq synthase [29], and pqqC was often used as an important molecular marker for soil inorganic phosphorus bacteria [30]. The results of qPCR showed that the absolute abundance of phoD and pqqC in SMOF treatment was significantly higher than in CF treatment (p < 0.05) by 760% and 263%, respectively (Figure 1c). Therefore, SMOF could significantly promote P-related micro-organism growth.
Chao1 and Shannon indexes reflected the richness and diversity of micro-organisms, respectively. Compared with CF, Chao1 and Shannon indexes in SMOF treatment increased significantly by 31% and 11%, respectively (p < 0.05) (Figure 1d). It might be attributed to livestock manure organic fertilizer to effectively regulate soil moisture and nutrient conditions. Furthermore, it provided more organic matter and other nutrients, reducing competition among bacteria and increasing bacterial richness and diversity [31,32]. The higher diversity in SMOF was beneficial to enhancing the stability of soil microbial community structure and soil function [33].

3.2. Soil pH, Bulk Density, Soil Enzyme Activity, and Soil Nutrient Content

Soil bulk density, as a critical parameter for evaluating soil porosity and compactness, directly reflected soil aeration and permeability performance. Soil pH was key to soil nutrient efficiency, microbial community, and crop growth [4]. Low pH would lead to low nutrient uptake efficiency by crops and microbial dysfunction [34]. The result showed that long-term SMOF significantly decreased soil bulk weight by 8% (p < 0.05) and significantly increased soil pH by 8% (p < 0.05) (Figure 2), which was consistent with results of the previous studies [4,35]. The improvement of soil pH, soil aeration, and permeability enhanced microbe activity and crop growth [4,34,35].
Soil enzyme activities, as key indicators for assessing microbial activity and soil biochemical reactions, played a crucial role in driving nutrient cycling and metabolic processes in soil ecosystems [35]. The result showed that, compared with CF treatment, the activities of soil Ure, ALP, and Suc in SMOF treatment increased significantly (p < 0.05) by 56%, 562%, and 160%, respectively (Figure 3) (p < 0.05). It might be attributed to rich nutrients in SMOF and the improved soil pH and bulk density, which promoted bacteria reproduction and viability and increased soil enzymes activity [12,34].
The result showed that long-term SMOF significantly increased soil nutrient content (p < 0.05). Compared with CF treatment, the contents of SOC, TN, AN, and AP in SMOF increased by 47%, 36%, 50%, and 29%, respectively (Figure 4a–d). The higher soil nutrient contents in SMOF treatment might be, firstly, attributed to long-term SMOF significantly lowering soil bulk density and increasing soil pH, which effectively improved soil aeration and permeability properties, alleviating soil acidification and, therefore, promoted the nutrient cycle [4,34,35]. Secondly, it might be attributed to the increase in relative abundance of such beneficial bacteria as Proteobacteria and Bacteroidota, and the absolute abundance of P-related functional genes such as pqqC and phoD for sheep manure organic fertilizer substitution. Pearson correlation coefficient analysis revealed that the relative abundance of Proteobacteria was significantly positively correlated with the contents of soil TN, soil AN and AP contents, and ALP activity (p < 0.05). The relative abundance of Bacteroidota was extremely significantly positively correlated with ALP activity, soil AP, and TN (p < 0.01). The absolute abundance of P-related functional genes phoD and pqqC was extremely significantly positively correlated with ALP activity, Suc activity, and AP content (p < 0.01), and significantly positively correlated with Ure activity, soil TN, and AN contents (p < 0.05) (Figure 4e). The increased abundance of P-involved functional genes and beneficial bacteria with the capacity of fixing nitrogen and solubilizing or mineralizing P strengthened the cycling of such soil nutrients as nitrogen and P [17,27,30]. Thirdly, it might be attributed to the significant increase in soil enzyme activities in SMOF treatment. Pearson correlation coefficient analysis showed that soil ALP activity was extremely significantly positively correlated with soil AP content, and soil Ure activity was significantly positively correlated with soil TN and AN content (Figure 4e). The increase in soil enzyme activity, such as ALP and Ure, in SMOF treatment enhanced the mineralization of soil organic P and nitrogen and soil nutrient cycling [26,27].

3.3. MDA Content and Antioxidant Enzyme Activity in Strawberry Leaves

The results showed that SMOF significantly reduced MDA content and increased antioxidant enzyme activity in strawberry leaves (p < 0.05) (Figure 5). Compared with CF treatment, MDA content in SMOF treatment decreased by 53% and the activities of SOD, CAT, and POD in SMOF treatment increased by 48%, 54%, and 58%, respectively (Figure 5), which was beneficial to efficiently enhancing strawberry resilience, fruit yield, and quality.

3.4. Strawberry Photosynthetic Pigment Content and Plant Biomass

The result showed that SMOF significantly increased strawberry photosynthetic pigment content and plant biomass (Figure 6). Compared with CF treatment, Chl a, Chl b, and Car contents in SMOF treatment increased significantly by 11%, 19%, and 11%, respectively (p < 0.05). Strawberry shoot biomass in SMOF treatment increased significantly by 25% (p < 0.05), and root biomass increased insignificantly (p > 0.05). It might be attributed to the content increase in such soil nutrients as nitrogen and P caused by SMOF application (Figure 4) and enhanced strawberry nutrient uptake and photosynthesis [36]. Nitrogen and P played an important role in the synthesis of photosynthetic pigments, and the increase in nitrogen and P content enhanced strawberry photosynthesis and biomass accumulation [37,38].

3.5. Strawberry Fruit Nutritional Quality and Yield

The result showed that compared with CF treatment, the strawberry fruit yield in SMOF treatment increased significantly by 40% (p < 0.05). The contents of strawberry fruit TSS, SS, SP, and Vc and sugar–acid ratio in SMOF treatment were significantly higher than in CF treatment by 21%, 15%, 46%, 23%, and 41%, respectively (p < 0.05) (Figure 7).
This study revealed that long-term SMOF significantly increased strawberry fruit yield and nutritional quality (p < 0.05) (Figure 7). The reasons were as follows: Firstly, increasing soil bacterial diversity and the abundance of beneficial bacteria and P-related functional genes (pqqC and phoD) might be attributed to SMOF. Chao1 and Shannon indexes in SMOF treatment were higher than those in CF treatment by 31% and 11%, respectively. Higher microbial diversity contributed to more complete and stable soil ecosystem function. In addition, higher beneficial bacteria abundance promoted the nutrition cycle. The increase in Chloroflexi abundance in SMOF treatment promoted carbon and nitrogen cycling. Higher abundance of beneficial bacteria such as Proteobacteria and Bacteroidota, and P-related functional genes pqqC and phoD, in SMOF treatment promoted nitrogen and P cycling, which was beneficial to the improvement of strawberry fruit yield and nutritional quality.
Notably, this might be due to the enhanced synergistic action among different bacteria for SMOF application. In order to explore interaction among soil bacteria in different fertilizer applications, co-occurrence network analysis was performed based on the Spearman correlation of the top 50 genera with the highest relative abundance (Figure 8a,b). The co-occurrence network analysis indicated that in SMOF treatment, the positive relationships were more than the negative ones and accounted for 59%, but the opposite was the case in CF treatment (Figure 8a,b). In CF treatment, the positive correlation among bacteria only accounted for 49%. The dominant positive relationships in the network in SMOF treatment suggested enhanced synergistic action among bacteria in an ecosystem function such as material cycling [39]. In SMOF treatment, the abundance of beneficial bacteria such as Proteobacteria and Bacteroidota, and P-related function genes pqqC and phoD, increased and provided more soil available phosphorus and nitrogen for other bacteria such as Chloroflexi. The increase in Chloroflexi abundance might enhance soil sucrase activity and provide more available carbon for other bacteria by accelerating organic matter decomposition and reducing bacteria competition [40,41]. The enhanced synergistic action in SMOF improved ecosystem function and stability, and was beneficial to strawberry yield and nutritional quality.
Thirdly, it might be attributed to an improvement in soil physicochemical properties by SMOF, such as lower soil bulk density and higher pH, soil enzyme activity, and soil nutrient content. Pearson correlation analysis revealed that strawberry fruit yield and nutritional quality such as the contents of SS, SP, TSS, and Vc were negatively correlated with soil bulk density and positively with soil pH, enzyme activity, and the contents of soil TN, AN, and AP (Figure 8c). Lower soil bulk density caused by SMOF increased soil porosity, and improved soil water and air permeability, which provided good growing conditions for strawberry roots and rhizospheric bacteria. Soil acidification alleviation in SMOF treatment effectively enhanced ammonification and increased AN content [42,43], which promoted nitrogen uptake by strawberries, and improved strawberry fruit yield and nutritional quality. In addition, long-term application of sheep manure organic fertilizer significantly increased soil AP content and P uptake by strawberry. It had been demonstrated that P played an indispensable role in photosynthesis and in the formation of soluble sugar and nutrient transformation [44]. Higher P content could decrease total fruit acidity and increase SS content, sugar–acid ratio, and Vc content in fruit [45].
Fourthly, it might be attributed to the enhancement of strawberry antioxidant capacity and photosynthesis by long-term application of sheep manure organic fertilizer. Compared with CF treatment, MDA content in SMOF treatment decreased by 53% and the activities of SOD, CAT, and POD increased by 48%, 54%, and 58%, respectively. Higher activities of antioxidant enzymes in SMOF treatment contributed to eliminating more reactive oxygen species and alleviating membrane lipid peroxidation damage to maintain cellular homeostasis, which was beneficial to the improvement of strawberry fruit yield and nutritional quality. In addition, the contents of Chl and Car in strawberry leaves in SMOF treatment were significantly higher than in the CF treatment. It was conducive to the enhancement of photosynthesis and the promotion of sugar accumulation and protein synthesis. The improved strawberry rhizospheric health by SMOF enhanced strawberry fruit yield and nutritional quality (Figure 8d).

4. Conclusions

This study revealed that long-term SMOF substitution for CF could efficiently improve strawberry performance and rhizospheric health. In SMOF treatment, soil nutrient content, pH value, enzyme activity, absolute abundance of P-related functional genes (pqqC and phoD), and bacteria diversity increased significantly after 16 year’s of SMOF application. Co-occurrence network analysis found that long-term SMOF application enhanced synergistic action among strawberry rhizospheric bacteria. Pearson correlation coefficient analysis showed that strawberry fruit yield and nutritional quality were positive with soil enzyme activity, soil nutrient content, and bacterial diversity indices. After 16 year’s of SMOF application, strawberry plant growth, fruit yield, and nutritional quality were enhanced by strawberry rhizospheric health.

Author Contributions

Conceptualization, Z.G. and Y.L.; methodology, Y.L.; software, D.Z., S.G. and J.W.; validation, D.Z.; formal analysis, D.Z.; investigation, Z.C. and C.L.; resources, Z.G.; data curation, X.C.; writing—original draft preparation, Z.C., C.L., X.C. and Z.G.; writing—review and editing, Z.C., Y.L., S.G., J.W. and Z.G.; visualization, S.G.; supervision, Z.G.; project administration, Z.G.; funding acquisition, Z.G. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the Key Project of Xihua University (Z212040). The APC was funded by the Key Project of Xihua University (Z212040).

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author(s).

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Wang, C.; Liu, D.W.; Bai, E. Decreasing soil microbial diversity is associated with decreasing microbial biomass under nitrogen addition. Soil Biol. Biochem. 2018, 120, 126–133. [Google Scholar] [CrossRef]
  2. Hao, W.; Wang, X.; Wang, Y.; Ma, J.; Tan, J. Harm of excessive fertilization on wheat and ways of reducing fertilizer application. Mod. Agric. Sci. Technol. 2022, 24, 40–44. [Google Scholar]
  3. Du, Y.D.; Cui, B.J.; Zhang, Q.; Wang, Z.; Sun, J.; Niu, W.Q. Effects of manure fertilizer on crop yield and soil properties in China: A meta-analysis. Catena 2020, 193, 104617. [Google Scholar] [CrossRef]
  4. Liu, Y.; Xu, M.; Liu, M.J.; Cheng, C.A.; Qiao, L.; Li, Y.Q.; Li, Z. Responses of soil available nutrients and microbial performance in a newly established apple orchard after five-year fertilization with different sources of livestock manure. J. Agric. Food Res. 2025, 19, 101635. [Google Scholar] [CrossRef]
  5. Kai, T.; Adhikari, D. Effect of organic and chemical fertilizer application on apple nutrient content and orchard soil condition. Agriculture 2021, 11, 340. [Google Scholar] [CrossRef]
  6. Wang, L.Y.; Zhang, Y.C.; Chen, L.L.; Li, R.N.; Zhai, C.X.; Li, Y.Q. Effects of different kinds of manure combination with chemical fertilizer on yield, quality and soil nutrient content in greenhouse tomato. Acta Agric. Boreali-Sin. 2011, 26, 152–156. [Google Scholar]
  7. Li, P.; Kong, D.N.; Zhang, H.J.; Xu, L.Y.; Li, C.K.; Wu, M.C.; Jiao, J.G.; Li, D.M.; Xu, L.; Li, H.X.; et al. Different regulation of soil structure and resource chemistry under animal-and plant-derived organic fertilizers changed soil bacterial communities. Appl. Soil Ecol. 2021, 165, 104020. [Google Scholar] [CrossRef]
  8. Dinesh, R.; Srinivasan, V.; Hamza, S.; Manjusha, A. Short-term incorporation of organic manures and biofertilizers influences biochemical and microbial characteristics of soils under an annual crop [Turmeric (Curcuma longa L.)]. Bioresour. Technol. 2010, 101, 4697–4702. [Google Scholar] [CrossRef] [PubMed]
  9. Food and Agriculture Organization of the United Nations. Crop Sand Livestock Products. 2023. Available online: https://www.fao.org/faostat/en/#data/QCL (accessed on 20 February 2023).
  10. Nakielska, M.; Berbec, A.K.; Madej, A.; Feledyn-Szewczyk, B. Microbial Fertilizing Products Impact on Productivity and Profitability of Organic Strawberry Cultivars. Horticulturae 2024, 10, 1112. [Google Scholar] [CrossRef]
  11. Vishwakarma, G.; Shukla, A.K.; Zaman, F.; Singh, A.; Shukla, S.K. Assessment of Strawberry to Integrated Nutrient Management for Different Yield Attributes and Quality Parameters. Appl. Fruit Sci. 2024, 66, 833–841. [Google Scholar] [CrossRef]
  12. Zha, Y.; Liu, A.C.; Lai, W.G.; Wang, J.R.; Li, X.Y.; Yu, H.; Xiao, W.F. Sheep manure organic fertilizer is an effective strategy to promote strawberry growth by improving soil physicochemical properties and microbiota. Front. Environ. Sci. 2024, 12, 1414010. [Google Scholar] [CrossRef]
  13. Quddus, M.A.; Ahmed, R.; Islam, M.; Haque, M.E.; Islam, M.A.; Alam, A.; Rahman, M.Z.; Fahad, Z.H.; Islam, M.K.; Gaber, A.; et al. Organic and inorganic fertilizers influence the productivity, fruit quality and nutrient use efficiency of strawberry (Fragaria × ananassa Duch.). Sci. Rep. 2025, 15, 26252. [Google Scholar] [CrossRef]
  14. Yang, J.H.; Wang, C.L.; Dai, H.L. Soil Agricultural Chemistry Analysis and Environmental Monitoring, 1st ed.; China Land Press: Beijing, China, 2008; pp. 18–20. [Google Scholar]
  15. Lu, R.K. Methods of Soil Agricultural Chemical Analysis, 2nd ed.; China Agricultural Science and Technology Press: Beijing, China, 2000; pp. 12–185. [Google Scholar]
  16. Guan, S.Y. Soil Enzymes and Their Research Methods, 1st ed.; Agriculture Press: Beijing, China, 1986; pp. 260–334. [Google Scholar]
  17. Li, H.L.; Zhu, H.T.; Li, H.B.; Zhang, Y.Q.; Xu, S.X.; Cai, S.M.; Sulaiman, A.A.; Kuzyakov, Y.; Rengel, Z.; Zhang, D.S. Dynamics of root-microbe interactions governing crop phosphorus acquisition after straw amendment. Soil Biol. Biochem. 2023, 181, 109039. [Google Scholar] [CrossRef]
  18. Li, H.S. Principles and Techniques of Plant Physiological and Biochemical Experiments, 1st ed.; Higher Education Press: Beijing, China, 2000; pp. 134–248. [Google Scholar]
  19. Zhang, X.; Li, J.; Shao, L.; Qin, F.; Yang, J.; Gu, H.R.; Zhai, P.; Pan, X.Q. Effects of organic fertilizers on yield, soil physico-chemical property, soil microbial community diversity and structure of Brassica rapa var. Chinensis. Front. Microbiol. 2023, 14, 1132853. [Google Scholar] [CrossRef]
  20. Wan, J.X.; Wang, X.F.; Yang, T.J.; Wei, Z.; Banerjee, S.; Friman, V.P.; Mei, X.L.; Xu, Y.C.; Shen, Q.R. Livestock manure type affects microbial community composition and assembly during composting. Front. Microbiol. 2021, 12, 621126. [Google Scholar] [CrossRef]
  21. Azene, B.; Zhu, R.H.; Pan, K.W.; Sun, X.M.; Nigussie, Y.; Gruba, P.; Raza, A.; Guadie, A.; Wu, X.G.; Zhang, L. Land use change alters phosphatase enzyme activity and phosphatase-harboring microbial abundance in the subalpine ecosystem of southeastern Qinghai-Tibet Plateau, China. Ecol. Indic. 2023, 153, 110416. [Google Scholar] [CrossRef]
  22. Hu, M.J.; Penuelas, J.; Sardans, J.; Tong, C.; Chang, C.T.; Cao, W.Z. Dynamics of phosphorus speciation and the phoD phosphatase gene community in the rhizosphere and bulk soil along an estuarine freshwater-oligohaline gradient. Geoderma 2020, 365, 114236. [Google Scholar] [CrossRef]
  23. Shih, P.M.; Ward, L.M.; Fischer, W.W. Evolution of the 3-hydroxypropionate bicycle and recent transfer of anoxygenic photosynthesis into the Chloroflexi. Proc. Natl. Acad. Sci. USA 2017, 114, 10749–10754. [Google Scholar] [CrossRef]
  24. Qin, S.M.; Zhang, H.Y.; He, Y.H.; Chen, Z.J.; Yao, L.G.; Han, H. Improving radish phosphorus utilization efficiency and inhibiting Cd and Pb uptake by using heavy metal-immobilizing and phosphate-solubilizing bacteria. Sci. Total Environ. 2023, 868, 161685. [Google Scholar] [CrossRef] [PubMed]
  25. Wei, X.M.; Hu, Y.J.; Cai, G.; Yao, H.Y.; Ye, J.; Sun, Q.; Veresoglou, S.D.; Li, Y.Y.; Zhu, Z.K.; Guggenberger, G.; et al. Organic phosphorus availability shapes the diversity of phoD-harboring bacteria in agricultural soil. Soil Biol. Biochem. 2021, 161, 108364. [Google Scholar] [CrossRef]
  26. Cao, N.; Zhi, M.L.; Zhao, W.Q.; Pang, J.Y.; Hu, W.; Zhou, Z.G.; Meng, Y.L. Straw retention combined with phosphorus fertilizer promotes soil phosphorus availability by enhancing soil P-related enzymes and the abundance of phoC and phoD genes. Soil Tillage Res. 2022, 220, 105390. [Google Scholar] [CrossRef]
  27. Luo, G.W.; Sun, B.; Li, L.; Li, M.H.; Liu, M.Q.; Zhu, Y.Y.; Guo, S.W.; Ling, N.; Shen, Q.R. Understanding how long-term organic amendments increase soil phosphatase activities: Insight into phoD- and phoC-harboring functional microbial populations. Soil Biol. Biochem. 2019, 139, 107632. [Google Scholar] [CrossRef]
  28. Zheng, B.X.; Hao, X.L.; Ding, K.; Zhou, G.W.; Chen, Q.L.; Zhang, J.B.; Zhu, Y.G. Long-term nitrogen fertilization decreased the abundance of inorganic phosphate solubilizing bacteria in an alkaline soil. Sci. Rep. 2017, 7, 42284. [Google Scholar] [CrossRef]
  29. Meyer, J.B.; Frapolli, M.; Keel, C.; Maurhofer, M. Pyrroloquinoline quinone biosynthesis gene pqqC, a novel molecular marker for studying the phylogeny and diversity of phosphate-solubilizing pseudomonads. Appl. Environ. Microbiol. 2011, 77, 7345–7354. [Google Scholar] [CrossRef]
  30. Long, X.E.; Yao, H.Y.; Huang, Y.; Wei, W.X.; 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]
  31. Chen, Y.; Sun, R.B.; Sun, T.T.; Liang, Y.T.; Jiang, Y.J.; Sun, B. Organic amendments shift the phosphorus-correlated microbial co-occurrence pattern in the peanut rhizosphere network during long-term fertilization regimes. Appl. Soil Ecol. 2018, 124, 229–239. [Google Scholar] [CrossRef]
  32. Li, X.Q.; Lu, Q.J.; Li, D.Y.; Wang, D.Z.; Ren, X.X.; Yan, J.L.; Ahmed, T.; Li, B. Effects of Two Kinds of commercial organic fertilizers on growth and rhizosphere soil properties of corn on new reclamation land. Plants 2022, 11, 2553. [Google Scholar] [CrossRef] [PubMed]
  33. Chaer, G.; Fernandes, M.; Myrold, D.; Bottomley, P. Comparative resistance and resilience of soil microbial communities and enzyme activities in adjacent native forest and agricultural soils. Microb. Ecol. 2009, 58, 414–424. [Google Scholar] [CrossRef] [PubMed]
  34. Jia, X.L.; Wang, Y.H.; Zhang, Q.; Lin, S.X.; Zhang, Y.; Du, M.R.; Chen, M.H.; Ye, J.H.; Wu, Z.Y.; Wang, H.B. Reasonable deep application of sheep manure fertilizer to alleviate soil acidification to improve tea yield and quality. Front. Front. Plant Sci. 2023, 14, 1179960. [Google Scholar] [CrossRef] [PubMed]
  35. Yin, H.K.; Li, Q.; Li, Y.H.; Xie, W.Y.; Zhou, H.P.; Yang, Z.X.; Liu, Z.P.; He, L.Y. Effects of different fertilization measures on microbial community and enzyme activities in brown soil. Environ. Sci. 2025, 1–20. [Google Scholar]
  36. Sahana, B.J.; Madaiah, D.; Shivakumar, B.S.; Sridhara, S.; Pradeep, S. Influence of organic manures on growth, yield and quality of strawberry (Fragaria × ananassa Duch.) under naturally ventilated polyhouse. J. Pharmacogn. Phytochem. 2020, 9, 3284–3287. [Google Scholar]
  37. Meng, Q.Q.; Shi, Z.J.; Yan, Z.B.; Lambers, H.; Luo, Y.; Han, W.X. Independent and interactive effects of N and P additions on foliar P fractions in evergreen forests of southern China. For. Ecosyst. 2025, 12, 100265. [Google Scholar] [CrossRef]
  38. Lambers, H. Phosphorus acquisition and utilization in plants. Annu. Rev. Plant Biol. 2022, 73, 17–42. [Google Scholar] [CrossRef]
  39. Yu, X.T.; Li, Y.; Wu, Y.; Gao, H.; Liu, W.; Liu, H.; Gong, S.D.; Wu, H.L. Seasonal changes of prokaryotic microbial community structure in Zhangjiayan Reservoir and its response to environmental factors. Sci. Rep. 2024, 14, 5513. [Google Scholar] [CrossRef]
  40. Chaparro, J.M.; Badri, D.V.; Vivanco, J.M. Rhizosphere microbiome assemblage is affected by plant development. ISME J. 2014, 8, 790–803. [Google Scholar] [CrossRef]
  41. Hou, Q.; Wang, W.X.; Yang, Y.; Hu, J.; Bian, C.S.; Jin, L.P.; Li, G.C.; Xiong, X.Y. Rhizosphere microbial diversity and community dynamics during potato cultivation. Eur. J. Soil Biol. 2020, 98, 103176. [Google Scholar] [CrossRef]
  42. Scarlett, K.; Denman, S.; Clark, D.R.; Forster, J.; Vanguelova, E.; Brown, N.; Whitby, C. Relationships between nitrogen cycling microbial community abundance and composition reveal the indirect effect of soil pH on oak decline. ISME J. 2021, 15, 623–635. [Google Scholar] [CrossRef] [PubMed]
  43. Stopnisek, N.; Gubry-Rangin, C.; Höfferle, S.; Nicol, G.W.; Mandic-Mulec, I.; Prosser, J.I. Thaumarchaeal ammonia oxidation in an acidic forest peat soil is not influenced by ammonium amendment. Appl. Environ. Microbiol. 2010, 76, 7626–7634. [Google Scholar] [CrossRef]
  44. Madhavi, B.G.K.; Khan, F.; Bhujel, A.; Jaihuni, M.; Kim, N.E.; Moon, B.E.; Kim, H.T. Influence of different growing media on the growth and development of strawberry plants. Heliyon 2021, 7, e07170. [Google Scholar] [CrossRef] [PubMed]
  45. Wang, C.L.; Qu, M.S.; Zhao, Y.Z.; Zhou, J.Z.; Lei, W.W.; Chen, J.; Liu, Y.; Zhao, K.L. Effects of phosphorus fertilizer application rate on the yield and quality of protected strawberries. China Agric. Technol. Ext. 2018, 34, 49–51. [Google Scholar]
Horticulturae 11 01000 g001
Figure 1. Microbial community structure and P-related functional gene abundance in strawberry rhizospheric soil. (a,b) relative abundance of soil micro-organisms at phylum level (top 20) and at genus level (top 20); (c) absolute abundance of P-related functional genes (phoD and pqqC); and (d) α-diversity indices of strawberry rhizospheric soil micro-organisms. Different lower case letters on the bar graphs indicate significant differences between treatments (n = 3, p < 0.05).
Figure 1. Microbial community structure and P-related functional gene abundance in strawberry rhizospheric soil. (a,b) relative abundance of soil micro-organisms at phylum level (top 20) and at genus level (top 20); (c) absolute abundance of P-related functional genes (phoD and pqqC); and (d) α-diversity indices of strawberry rhizospheric soil micro-organisms. Different lower case letters on the bar graphs indicate significant differences between treatments (n = 3, p < 0.05).
Horticulturae 11 01000 g001
Horticulturae 11 01000 g002
Figure 2. Soil pH (a) and soil bulk density (b). Different lower case letters above the bar indicate significant differences between treatments (n = 3, p < 0.05).
Figure 2. Soil pH (a) and soil bulk density (b). Different lower case letters above the bar indicate significant differences between treatments (n = 3, p < 0.05).
Horticulturae 11 01000 g002
Horticulturae 11 01000 g003
Figure 3. Soil enzyme activity. (a) urease activity, (b) alkaline phosphatase activity, and (c) sucrase activity. Different lowercase letters above the bar indicate significant differences between treatments (n = 3, p < 0.05).
Figure 3. Soil enzyme activity. (a) urease activity, (b) alkaline phosphatase activity, and (c) sucrase activity. Different lowercase letters above the bar indicate significant differences between treatments (n = 3, p < 0.05).
Horticulturae 11 01000 g003
Horticulturae 11 01000 g004
Figure 4. The contents of (a) soil alkali-hydrolyzable nitrogen, (b) total nitrogen, (c) organic carbon, (d) available phosphorus, and (e) Pearson correlation analysis of soil nutrient content with soil enzyme activities, relative abundance of strawberry rhizobacteria, and absolute abundance of functional genes for phosphorus solubilization. Pro: Proteobacteria; Fir: Firmicutes; Chl: Chloroflexi; Aci: Acidobacteriota; Act: Actinobacteriota; Bac: Bacteroidota; Gem: Gemmatimonadota. Different lowercase letters above the bar indicate significant differences between treatments (n = 3, p < 0.05).
Figure 4. The contents of (a) soil alkali-hydrolyzable nitrogen, (b) total nitrogen, (c) organic carbon, (d) available phosphorus, and (e) Pearson correlation analysis of soil nutrient content with soil enzyme activities, relative abundance of strawberry rhizobacteria, and absolute abundance of functional genes for phosphorus solubilization. Pro: Proteobacteria; Fir: Firmicutes; Chl: Chloroflexi; Aci: Acidobacteriota; Act: Actinobacteriota; Bac: Bacteroidota; Gem: Gemmatimonadota. Different lowercase letters above the bar indicate significant differences between treatments (n = 3, p < 0.05).
Horticulturae 11 01000 g004
Horticulturae 11 01000 g005
Figure 5. MDA content and antioxidant enzyme activity in strawberry leaves. (a) malondialdehyde content; (b) superoxide dismutase activity; (c) catalase activity; and (d) peroxidase activity. Different lowercase letters above the bar indicate significant differences between treatments (n = 3, p < 0.05).
Figure 5. MDA content and antioxidant enzyme activity in strawberry leaves. (a) malondialdehyde content; (b) superoxide dismutase activity; (c) catalase activity; and (d) peroxidase activity. Different lowercase letters above the bar indicate significant differences between treatments (n = 3, p < 0.05).
Horticulturae 11 01000 g005
Horticulturae 11 01000 g006
Figure 6. (a) Photosynthetic pigment content and (b) plant biomass. Different lowercase letters above the bar indicate significant differences between treatments (n = 3, p < 0.05).
Figure 6. (a) Photosynthetic pigment content and (b) plant biomass. Different lowercase letters above the bar indicate significant differences between treatments (n = 3, p < 0.05).
Horticulturae 11 01000 g006
Horticulturae 11 01000 g007
Figure 7. Strawberry fruit nutritional quality and yield. (a) total soluble solids and soluble protein content, (b) soluble sugar and titratable acid content, (c) vitamin C content and sugar–acid ratio, and (d) strawberry fruit yield. Different lowercase letters above the bar indicate significant differences between treatments (n = 3, p < 0.05).
Figure 7. Strawberry fruit nutritional quality and yield. (a) total soluble solids and soluble protein content, (b) soluble sugar and titratable acid content, (c) vitamin C content and sugar–acid ratio, and (d) strawberry fruit yield. Different lowercase letters above the bar indicate significant differences between treatments (n = 3, p < 0.05).
Horticulturae 11 01000 g007
Horticulturae 11 01000 g008
Figure 8. Co-occurrence network analysis of rhizospheric bacteria in (a) CF treatment and (b) SMOF treatment. The node in network represented the genera (top 50) and the color of node represented different phyla. (c) the links of strawberry fruit yield and nutritional quality with leaf physiological properties, soil nutrient contents, enzyme activities, and bacteria diversity by Pearson correlation coefficient analysis. (d) schematic diagram of SMOF improving strawberry fruit yield and nutritional quality.
Figure 8. Co-occurrence network analysis of rhizospheric bacteria in (a) CF treatment and (b) SMOF treatment. The node in network represented the genera (top 50) and the color of node represented different phyla. (c) the links of strawberry fruit yield and nutritional quality with leaf physiological properties, soil nutrient contents, enzyme activities, and bacteria diversity by Pearson correlation coefficient analysis. (d) schematic diagram of SMOF improving strawberry fruit yield and nutritional quality.
Horticulturae 11 01000 g008
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

Chou, Z.; Lei, C.; Cai, X.; Li, Y.; Zeng, D.; Gong, S.; Wang, J.; Gong, Z. Strawberry Performance and Rhizospheric Health Were Efficiently Improved After Long-Term Sheep Manure Organic Fertilizer Application. Horticulturae 2025, 11, 1000. https://doi.org/10.3390/horticulturae11091000

AMA Style

Chou Z, Lei C, Cai X, Li Y, Zeng D, Gong S, Wang J, Gong Z. Strawberry Performance and Rhizospheric Health Were Efficiently Improved After Long-Term Sheep Manure Organic Fertilizer Application. Horticulturae. 2025; 11(9):1000. https://doi.org/10.3390/horticulturae11091000

Chicago/Turabian Style

Chou, Zhengyan, Chenghao Lei, Xinyi Cai, Yong Li, Diya Zeng, Sidan Gong, Jianping Wang, and Zhilian Gong. 2025. "Strawberry Performance and Rhizospheric Health Were Efficiently Improved After Long-Term Sheep Manure Organic Fertilizer Application" Horticulturae 11, no. 9: 1000. https://doi.org/10.3390/horticulturae11091000

APA Style

Chou, Z., Lei, C., Cai, X., Li, Y., Zeng, D., Gong, S., Wang, J., & Gong, Z. (2025). Strawberry Performance and Rhizospheric Health Were Efficiently Improved After Long-Term Sheep Manure Organic Fertilizer Application. Horticulturae, 11(9), 1000. https://doi.org/10.3390/horticulturae11091000

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