Two treatments were carried out in this study. Treatment one included the use of coconut bran, provided by Xiamen Huanshi Import and Export Trading Co., Ltd., as the substrate. After washing, the electrical conductivity (EC) of the coconut bran was 0.7–0.9 ms/cm. In treatment two, the substrate comprised coconut bran and vermicompost in a ratio of 16:1, and the vermicompost was fermented using rice husk and cow dung as carriers. Both treatments were repeated three times, and each planting area was considered one plot with a length of 10 m, depth of 35 cm, and width of 30 cm. On 11 September 2021, “Zhangji” strawberry cultivar seedlings (provided by Kunming Kusen agricultural development Co., Ltd.) with three leaves and one bud were transplanted. A “W” type double-row planting, with a row spacing of 20 cm × 20 cm, was followed, and approximately 100 healthy “Zhangji” seedlings were transplanted into each plot (N36°05′53.31″, E116°57′53.16″). To enhance the reproducibility of the study, the agricultural procedures of the two treatments were kept consistent. After sampling for measuring substrate nutrients, a large amount of elemental water-soluble fertilizer containing N, P, and K content of 20% each was applied. Every 15–20 days, 50 g of the fertilizer was wash-applied per implant until the end of the harvesting period.

Forty days after the strawberry seedlings were transplanted, 15 strawberry plants were randomly selected from each plot. Their height and canopy were measured using a ruler, while their relative chlorophyll content and leaf area of the third developed leaf were determined using a SPAD-502 handheld chlorophyll analyzer (SPAD-502 +, Konica Minolta, Inc.) and a YMJ leaf area analyzer (CI-202, CID Bio-Science, Inc.), respectively. In addition, 30 days (growth period) and 120 days (fruiting period) after transplantation, three random strawberry plants from each plot were uprooted and rinsed thoroughly, and their surface was dried out. The aboveground and belowground parts of strawberry plants were separated and weighed separately to obtain measurements of their fresh weight (Shrestha et al., 2024).

The activities of soil enzymes, including cellulase, urease, and phosphatase, as well as plant nutrient contents, were measured following the established protocols (Li et al., 2020). Cellulase activity was quantified using the Grice Soil Cellulase Kit (G0308F), Suzhou, China. Urease activity was assessed using the conductivity method, whereas acid phosphatase activity was determined using the acetate method. In addition, nitrate content was analyzed via ultraviolet spectrophotometry. Alkali-hydrolyzable nitrogen content was measured using the alkali diffusion method. Furthermore, available-P content was determined through sodium bicarbonate extraction followed by the molybdenum–antimony anti-colorimetric method, and available-K content was assessed using ammonium acetate extraction and flame spectrophotometry. Organic matter content was quantified using the hydrated potassium dichromate oxidation colorimetric method. Moreover, cation exchange capacity (CEC) was determined using the single balance method.

Substrate sampling was carried out 40 days after strawberry seedling transplantation. First, 15 sampling points of approximately 5 cm from strawberry plants were randomly selected in each plot. After removing the surface matrix, the substrate was collected from a depth of 5–15 cm, thoroughly mixed, sieved through a 2 mm mesh, and divided into two parts. Part 1 was stored at 4°C to determine soil enzyme activity and physicochemical properties. Part 2 was freeze-dried with liquid nitrogen and stored in dry ice for high-throughput sequencing to analyze fungal and bacterial diversity. At the peak fruiting stage of 4 months after transplanting, substrate samples were taken again using the same method. The samples were thoroughly mixed, sieved through a 2 mm mesh, and divided into two parts, both of which were freeze-dried with liquid nitrogen. While part one was used for high-throughput sequencing to analyze fungal and bacterial diversity, part two was used for the transcriptome analysis of microbial substances. Sequencing technology was provided by Shanghai Lingen Biotechnology Co., Ltd.

At the mature stage of the first fruit (after 90–120 days), 15 near-mature fruit were randomly selected from individual plants of each plot, and single-fruit weight and soluble solid content were measured (Chiomento et al., 2021; Drobek et al., 2024). The fruit from each plot were harvested at maturity, and the cumulative yield was calculated. To facilitate operation, the area of the aisle was kept relatively large. The average yield of each plant was calculated based on 100 plants in each plot, and the equivalent yield was calculated based on 8,000 plants per 666.7 m².

The sample of vermicompost was collected, air-dried, and sifted using a 2 mm mesh. Then, 5.0 g of the sample was weighed and placed in a 100 mL triangle bottle, 45 mL of sterile water was added, and the bottle was swirled and oscillated for 10 min to obtain a 10⁻¹ sample suspension. Sample suspensions with concentrations of 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, and 10⁻⁶ were obtained by gradient dilution in sequence. The suspensions of 100 μL with a concentration of 10⁻⁴, 10⁻⁵, and 10⁻⁶ were placed on Luria–Bertani (LB) medium, coated evenly, and cultured in the dark at 33°C for 2 days. Single colonies were selected and cultured on a new LB medium plate for isolation and purification, and Fusarium oxysporum and Neopestalotiopsis sp. were used for the fungi antifungal test (Cheng et al., 2019; Wang et al., 2024). The two pathogens were provided by the provincial Key Laboratory of Agricultural Microbiology, Shandong Agricultural University.

The bacterial strain that had the best inhibitory effect (Wang et al., 2024) on F. oxysporum was selected from 15 antagonistic strains with potential biocontrol properties, and this strain was named JNS-1. The identity of the strain was determined based on multiple criteria, such as physiological and biochemical indicators according to 16S rRNA using primers 1492R/27F (1492R: 5’-GGTTACCTTGTTACGACTT-3′; 27F: 5’-AGAGTTGATCCTGGCTCAG-3′) (Wang et al., 2024). Strain morphology was observed using the LB medium in combination with a scanning electron microscope. The phylogenetic tree was constructed using the maximum likelihood method based on 16S rRNA regions performed in MEGA 7.0.

Strawberry seedlings of the “Zhangji” variety with three leaves and one core stage were transplanted separately in a plastic pot of 15 cm diameter with a special substrate for strawberries. The pots were further inoculated with conidial suspension (1 × 10⁶ spores/ml) of each pathogen, which was thoroughly mixed into the soil. For the treatment group that received liquid bactericide, 10 mL and 20 mL of the bactericide solution were injected into the rhizosphere of each strawberry plant, respectively, whereas, in the solid bactericide treatment group, 10 g and 20 g of the solid bactericide were applied to the rhizosphere of each plant, respectively. The control group remained untreated. Each treatment was conducted in triplicate, with 10 pots per replicate. The incidence of root rot disease was evaluated 15 and 30 days after treatment under standard cultivation conditions, and the relative control efficiency was subsequently calculated (Yang et al., 2024).

In the strawberry field where the “Zhangji” cultivar had been cultivated for 5 consecutive years, a severe incidence of root rot was observed. In the liquid inoculant treatment group, 10 mL and 20 mL of the liquid inoculant were poured into the rhizosphere of each strawberry plant, respectively, whereas in the solid inoculant treatment group, 10 g and 20 g of the solid inoculant were applied to the rhizosphere of each strawberry plant, respectively. Untreated strawberry seedlings were used as controls. Each treatment was carried out in triplicate, the study area was 10 m², and approximately 120 strawberry seedlings were planted. The incidence of strawberry root rot disease was investigated after 15 and 30 days, and the relative control efficiency was calculated (Yang et al., 2024).

Statistical analysis was conducted using SPSS 26.0 (IBM Corporation, Armonk, NY, United States). The Shapiro–Wilk test was used to evaluate the normality of the data, and the result showed that a p-value of greater than 0.05 indicates that the data matched the normal distribution. Independent samples t-tests were performed to compare the differences between treatments.

The addition of vermicompost to coconut coir significantly increased the activity of three soil enzymes, namely cellulase, urease, and acid phosphatase, whose activities were 0.94, 1.72, and 3.30 U, respectively, which were significantly higher than those in the coconut-coir-only treatment (0.69, 0.96, and 1.97 U, respectively) (p < 0.01) (Figure 1).

The addition of vermicompost significantly increased the nitrogen content of the strawberry-planted substrate. For example, NO₃-N and alkaline-N contents in the coconut coir + vermicompost treatment were 93.37 and 445.10 mg/kg, respectively, which were significantly higher than those in the coconut-coir-only treatment (16.93 and 314.60 mg/kg, respectively) (Figure 2). Meanwhile, the organic matter content, available-K, and CEC in the coconut coir + vermicompost treatment were 727.90 g/kg, 7338.87 mg/kg, and 11.58 cmol/kg, respectively, which were significantly higher than those in the coconut-coir-only treatment (590.90 g/kg, 4887.90 mg/kg, and 8.23 cmol/kg, respectively). However, vermicompost application did not have a significant effect on the available-P content of the substrate.

Vermicompost application effectively improved strawberry plant growth (Figures 3, 4). Forty days after transplanting, the relative chlorophyll content and leaf area of strawberry leaves in the coconut coir + vermicompost treatment were 46.68 and 6123.50 mm², respectively, which were significantly higher than those in the coconut-coir-only treatment (38.56 and 4895.10 mm², respectively). Meanwhile, 120 days after transplanting, the relative chlorophyll content and leaf area of strawberry leaves in the coconut coir + vermicompost treatment were 47.18 and 6088.93 mm², respectively, which were significantly higher than those in the coconut-coir-only treatment (40.80 and 5215.33 mm², respectively).

Thirty days after transplanting, the fresh weights of the aboveground and belowground parts of strawberry plants in the coconut coir + vermicompost treatment were 10.98 and 2.36 g, respectively, which were significantly higher than those in the coconut-coir-only treatment (8.83 and 1.93 g, respectively). With time, the difference in fresh weight between the two treatments gradually decreased; 120 days after transplanting, the fresh weights of aboveground and belowground parts did not significantly differ between the two treatments.

Vermicompost application transformed the microbial diversity of the substrate (Figure 5). A total of 10 bacterial phyla (Figure 5A), the most abundant of which was Proteobacteria, and 42 bacterial genera (Figure 5B), the most abundant of which was Devosia, were detected among the bacterial communities. Among the fungal communities, five fungal phyla (Figure 5C), the most abundant of which was Ascomycota, and 30 fungal genera (Figure 5D), the most abundant of which was Podospora, were detected. Moreover, vermicompost application increased the beneficial microbial transcriptome level (Figures 5, 6).

Vermicompost application advanced the average ripening time of the first strawberry crop by 7 days compared with the coconut-coir-only treatment. The single-fruit weight and soluble solid content of first-crop fruit in the coconut coir + vermicompost treatment were 25.40 g and 14.69%, respectively, which were significantly higher than those in the coconut-coir-only treatment (22.78 g and 13.71%, respectively) (Figure 7). The average plant yield of first-crop fruit in the coconut coir + vermicompost treatment was 63.20 g, which was significantly higher than the 47.00 g in the coconut-coir-only treatment. Moreover, the equivalent yield in the coconut coir + vermicompost treatment was 7.584 t/ha, which was significantly higher than the 5.640 t/ha in the coconut-coir-only treatment.

Among the 15 strains tested, JNS-1 showed the best antagonistic effect against Fusarium oxysporum, with an inhibitory zone rate of 59.2% (Figure 8). B. velezensis JNS-1 is preserved in the China General Microbiological Culture Collection Center under CGMCC no. 22531. In the 16S rRNA gene phylogenetic tree, JNS-1 (OR574161) showed a high bootstrap support value with B. velezensis FZB42 (NR075005), reaching 100% (Figure 9). As determined using scanning electron microscopy, B. velezensis JNS-1 was found to be rhabdoid and easy to aggregate to form biofilm (Figure 10).

According to the doses of 10 mL/plant and 20 mL/plant, the control effectiveness of JNS-1 liquid preparation on strawberry root rot disease caused by F. oxysporum was 80.00 and 100.00% at 15 days and 75.00% at 30 days, respectively. Furthermore, according to the dosage of 10 g/plant and 20 g/plant, the control efficiency of JNS-1 solid preparation on strawberry root rot disease caused by F. oxysporum was 80.00 and 90.00% at 15 days and 87.50% at 30 days, respectively.

According to the doses of 10 mL/plant and 20 mL/plant, the control effectiveness of JNS-1 liquid preparation on strawberry root rot disease caused by Neopestalotiopsis sp. was 66.67 and 77.78% at 15 days and 70.59 and 82.35% at 30 days, respectively. According to the dosage of 10 g/plant and 20 g/plant, the control effectiveness of JNS-1 solid preparation on strawberry root rot disease caused by Neopestalotiopsis sp. was 66.67 and 77.78% at 15 days and 70.59 and 82.35% at 30 days, respectively.

According to the doses of 10 mL/plant and 20 mL/plant, the control effectiveness of JNS-1 liquid preparation on strawberry root rot disease at 15 days was 75.00 and 87.96%, and the control effectiveness at 30 days was 78.17 and 85.91%, respectively. According to the dosage of 10 g/plant and 20 g/plant, the control effect of JNS-1 solid preparation on strawberry root rot disease at 15 days was 76.85 and 87.04%, and the control effect at 30 days was 78.87 and 85.21%, respectively.