An antagonistic microorganism was isolated from the rhizosphere soil of a healthy cucumber plant in cucumber-growing areas of the Nanjing Institute of Vegetable Science (118°46.615’E, 31°43.195’N) in Nanjing, Jiangsu Province, China. The soil suspension was serially diluted 10-fold, spread on Luria–Bertani (LB) medium, and incubated at 30°C for 3 days. Purified colonies were screened for their antagonism to fungal pathogen FOC on plates using the dual culture method (Rocha et al., 2017). The bacterial strain showing the strongest antifungal effect was coded as B2 and selected for further studies.

Pleurotus ostreatus strain P5 was isolated from spent P. ostreatus substrate (Wang et al., 2020). The fungal isolate was maintained on potato dextrose agar (PDA) slants at 4°C.

The pathogen used throughout this study was FOC [Agricultural Culture Collection of China (ACCC) No. 30220], which was provided by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. FOC was stored on PDA slants at 4°C.

The soil used in this experiment was collected from the surface layer of soil (0–15 cm) from a greenhouse at the Nanjing Institute of Vegetable Science. The soil at this site is classified as yellow-brown earth based on the Chinese soil classification and as Hapludalf, Alfisols based on the USDA’s soil classification (Wang et al., 2017). The studied greenhouse has continuously produced cucumbers for over 10 years. The collected soil was homogenized, passed through a 2-mm sieve at field moisture, and stored at 4°C until being used. The soil pH was 6.1; the organic carbon content was 11.3 g kg^–1; and the contents of total nitrogen, total phosphorus, and total potassium were 1.73 g kg^–1, 1.64 g kg^–1, and 17.6 g kg^–1, respectively. The soil contained p-hydroxybenzoic acid, vanillic acid, ferulic acid, p-coumaric acid, and benzoic acid at concentrations of 84.7, 36.4, 59.2, 21.6, and 28.5 μg/g, respectively (Figure 1). Soil phenolic acid extraction and detection were performed as follows.

Phenolic acids were extracted and detected by the method described by Chen et al. (2011). Briefly, 50 g of soil was extracted with 50 ml of 1 M NaOH and kept at 25°C for 24 h in the dark. Samples were then shaken vigorously for 30 min and centrifuged at 10,000 r min^–1 for 10 min. The 12 M HCl was used to adjust the pH of the supernatant to 2.5. The solutions were extracted three times with an equal volume of diethyl ether and dried in a rotary evaporator. The solvent was dissolved in 1 ml acetonitrile and filtered through 0.45-μm fiber filtration film. Extracts were analyzed using a Waters e2695 high-performance liquid chromatography (HPLC) (Milford, MA, United States), containing a YMC ODS-A column (4.6 mm × 250 mm, 5 μm). The standard phenolic acids (Shanghai Aladdin Biochemical Technology Co., Ltd.) used for HPLC were p-hydroxybenzoic acid, p-coumaric acid, ferulic acid, cinnamic acid, syringic acid, vanillic acid, vanillin, and benzoic acid. The phenolic acids in the extracts were separated using a gradient elution of solvent A acetic water (0.2%) and solvent B acetonitrile at 254 nm with a flow rate of 0.8 ml min^–1. The gradient elusion program was as follows: for 0–27 min, 0–10% solvent B; for 27–42 min, 10–15% solvent B; for 42–50 min, 15–30% solvent B; for 50–60 min, 30–50% solvent B; and for 60–70 min, 50–100% solvent B. The concentrations of phenolic acids were calculated from the standard curves. The presence of phenolic acid was confirmed by an ESI mass spectrometer AB SCIEX Triple Quad^TM 4500 using positive ion mode (AB SCIEX, Framingham, MA, United States).

The traditional physiological and biochemical characteristics of strain B2 were identified based on Bergey’s Manual of Systematic Bacteriology. Strain B2 was further identified through the analysis of its 16S rDNA and gyrB gene sequences. Briefly, the genomic DNA of the strain B2 was extracted using the bacterial DNA extraction kit (Omega, Germany) and stored at –20°C. The 16S rDNA was amplified with the bacterial universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) (Eden et al., 1991), and the gyrB gene was amplified with the specific primers UP1 (5′-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTY GA-3′) and UP2r (5′-AGCAGGGTACGGATGTGCGAGCCRT CNACRTCNGCRTCNGTCAT-3′) (Yamamoto and Harayama, 1995). The 20-μl PCR mixture contained 2 μl dNTP (2 mM), 2 μl MgCl₂ (25 mM), 1.0 μl of each primer (10 mM), 2.0 μl PCR buffer (10×), 1.0 μl template DNA, 0.2 μl Taq DNA polymerase (5 U), and 10.8 μl double-distilled (dd) H₂O. The thermocycling procedure involved an initial denaturation at 95°C for 3 min, followed by 35 cycles at 95°C for 1 min, 50°C for 45 s, 72°C for 2 min, and a final extension at 72°C for 10 min. The PCR products were then purified and sequenced by Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China). A sequence similarity analysis was performed using the NCBI BLAST program¹, and the phylogenetic tree was constructed by the neighbor-joining (NJ) method using MEGA-X.

To study the effects of different initial concentrations of mixture of phenolic acids [p-hydroxybenzoic acid, vanillic acid, ferulic acid, p-coumaric acid, benzoic acid (1/1/1/1/1, w/w)] on degradation, 2-ml inocula containing 1.2 mg L^–1 of mycelia were added to 50-ml mineral salt medium (MSM; KCl 0.5 g, K₂HPO₄ 1 g, KNO₃ 2 g, MgSO₄ 0.5 g, FeSO₄ 0.01 g, and distilled water 1,000 ml—all autoclaved at 121°C for 30 min) with 100–1,000 mg L^–1 mixture of phenolic acids as the sole carbon and energy source. All treatments were incubated at 28°C and 180 rpm for 96 h. Each treatment had three replicates, and no fungal inoculation was used as a control. The fungal mycelia in the culture were filtered and washed with acetonitrile three times and dried at 80°C until having a constant weight. Phenolic acids were extracted three times with diethyl ether, and the combined extracts were dried via rotary evaporator. The residues were dissolved in 1 ml acetonitrile, and the amounts of phenolic acids in the acetonitrile were determined with HPLC, as described in Section Soil.

The mixture of phenolic acid (400 mg L^–1; p-hydroxybenzoic acid 80 mg L^–1, vanillic acid 80 mg L^–1, ferulic acid 80 mg L^–1, p-coumaric acid 80 mg L^–1, benzoic acid 80 mg L^–1) was used to evaluate the degradation ability of strain P5 based on the fungal biomass, and the degradation rate was relatively high at this concentration. The inoculum size and culture conditions were consistent with those mentioned in Section Identification of Optimal Concentration for P. ostreatus P5 Degradation, and the flasks without strain P5 inoculation were used as control.

Kinetics of the phenolic acid degradation in liquid culture were determined by harvesting samples after 0, 12, 24, 36, 48, 60, 72, 84, and 96 h and freezing the samples at –80°C for later analysis. The fungal mycelia biomass and residual phenolic acid concentrations were detected as described above.

The strain B2 was cultivated in LB medium as described above. Cells were harvested by centrifugation, and cell pellets were resuspended twice with sterile distilled water. The cell concentration of strain B2 in the suspension was adjusted to 1 × 10⁸ CFU ml^–1 by diluting it with sterile distilled water. P. ostreatus strain P5 was grown for 5 days in PDA liquid medium at 28°C (180 rpm). The culture was centrifuged, and the obtained fungal mycelia were washed twice with sterile distilled water and were used as fungal inocula. Cucumber seeds (Cucumis sativus L., JinChun-No. 4, Tianjin Cucumber Research Centre) were surface sterilized with ethanol and 2% sodium hypochlorite (2 and 5 min, respectively), rinsed four times in sterilized distilled water, and then germinated in 90-mm glass Petri plates covered with moist filter paper in the dark at 30°C for 36 h.

After germination, the seeds were sown into nursery cups containing 300 g sterilized (121°C, 30 min) nursery soil. Plant seedlings (two true-leaf stage) were transplanted into plastic pots (15 cm diameter, 20 cm height) with 2 kg of soil. Three days before transplanting, the soil was inoculated with spores of FOC at concentrations of 1 × 10⁴ CFU g^–1 of soil. The pot experiment was designed in a randomized complete block design with four replications. The various treatments were as follows: (1) CK, without any microbial treatment; (2) B2, inoculation with strain B2; (3) P5, inoculation with strain P5; and (4) B2 + P5, co-inoculation with strain B2 and strain P5. One day before transplanting, wet fungal mycelia (20.0 g wet weight equivalent to 2.14 g dry fungal weight) were suspended in 50 ml water and mixed with 2 kg of soil. After transplanting, 10 ml of a water solution with strain B2 (10⁸ CFU ml^–1) was added around the root zone of the plant seedlings in the relevant treatment using a syringe. To maintain uniformity of nutrient supply in the four treatments, non-bacterial treatments received strain B2 inocula that had been autoclaved (121°C, 30 min), and non-fungal treatments also received strain P5 inocula that had been autoclaved (121°C, 30 min). Each treatment had four blocks with 12 pots in each block. One seedling was grown in each plastic pot. The pot experiment was conducted in a greenhouse located at the Nanjing Institute of Environmental Science, Nanjing, China. The temperature ranged from 21 to 28°C, and the relative humidity ranged from 64 to 83%.

After 60 days of transplanting, the cucumber seedlings were gently removed from the pot and shaken lightly to remove the soil loosely attached to the plant roots. The soil tightly adhering to the roots was collected and considered rhizosphere soil. The rhizosphere soil samples were divided into two subsamples: one was stored at –80°C for molecular studies and the other was stored at –20°C for phenolic acid analysis. Seedling infection by FOC was monitored daily, and the cumulative number of infected seedlings was also recorded. Disease incidence (DI) was defined as the percentage of infected seedlings over the total number of seedlings in each block and was assessed when the disease symptoms appeared (>20% of leaves wilted) (Cao et al., 2011). After harvesting, the plant height, root length, plant dry weight, and root dry weight were also measured.

Total soil DNA was extracted from 0.25 g of rhizosphere soil using the UltraClean Soil DNA Isolation Kit (Mo Bio Laboratories Inc., United States). The FOC-specific SCAR primer FocF3 5′-AAACGAGCCCGCTATTTGAG-3′ and FocR7 5′-TATTTCCTCCACATTGCCATG-3′ designed by Lievens et al. (2007) was used in a real-time PCR assay. The real-time PCR conditions were previously described by Cao et al. (2011). Real-time PCR was run in an ABI 7500 instrument (Applied Biosystems, United States). For quantification, standard curves were created with a 10-fold dilution series of plasmids (pMD-19T vector, Takara Bio) containing the PCR products from the amplification of FOC DNA with the primer FocF3/FocR7. All real-time PCRs were performed in triplicate, and ddH₂O was used as a negative control to replace the template.

The extraction and determination of soil phenolic acids were performed as described above.

All data were analyzed using SPSS 17.0 statistical software. Statistical significance was evaluated at the 95% level with Duncan’s test (p < 0.05). The data from the pot experiment followed a normal distribution (Shapiro–Wilk test) except for abundance of FOC. Pearson correlations were used for disease incidence and phenolic acid content with normal distribution, and Spearman rank correlations were used for disease incidence and FOC (R software Version 3.2.2).

The bacterial strain B2, an antagonist against FOC (Figure 2), was obtained from the rhizosphere soil of the cucumber. Physiological and biochemical characteristics are shown in Table 1. From these results, strain B2 was affiliated to the genus Bacillus. To further identify strain B2, 16S rDNA and gyrB genes were analyzed by sequencing. The 16S rDNA sequence of the strain B2 (GenBank accession number: MW308308) shared 99.8% identity with the 16S rDNA sequences of B. amyloliquefaciens (NR117946.1). The phylogenetic analysis of 16S rDNA sequences clearly showed that the strain B2 and B. amyloliquefaciens were clustered together (Figure 3A). Additionally, its gyrB gene sequence (GenBank accession number: MW316056) shared 99.8% identity with the gyrB gene sequences of B. amyloliquefaciens (KC439665.1). The phylogenetic analysis of gyrB gene sequences showed that the strain B2 and B. amyloliquefaciens were clustered together (Figure 3B). Thus, based on physiological and biochemical testing, and 16S rDNA and gyrB gene sequences analyses, strain B2 was identified as B. amyloliquefaciens.

Strain B2 was tested for the presence of a number of plant-beneficial traits. The results of the characterization analysis showed that strain B2 had the ability to secret IAA (Table 2). Moreover, strain B2 was found to be positive for phosphate solubilization, siderophore production, and biofilm formation (Table 2). The root surfaces of cucumber seedlings that were inoculated with strain B2 were examined using scanning electron microscopy (SEM) (Figure 4). Bacterial cells were easily visible on the root surface and strongly adhered to the root surface because the sample preparation for SEM analysis requires multiple treatments and exhaustive washes. High-resolution images also showed bacteria-forming biofilm structures on the root surfaces (Figure 4).

The ratios of mixture phenolic acid degradation and fungal biomass in different concentration cultures after 96 h of cultivation are shown in Figure 5. When the initial concentration was 400 mg L^–1 or lower, 100% of the mixture of phenolic acids was degraded. However, if the concentration was greater than 400 mg L^–1, the ratio of the mixture of phenolic acid degradation significantly decreased and only 5.9% could be removed at the concentration of 1,000 mg L^–1. Moreover, the highest mycelial biomass was also observed at 400 mg L^–1. Thus, 400 mg L^–1 was chosen for the following degradation experiments.

The kinetics of the mixture of five phenolic acid degradation and strain P5 growth in liquid medium are shown in Figure 6. In the control, natural degradation efficiencies of all five phenolic acids at 96 h of incubation were 2.7, 4.4, 7.2, 5.0, and 4.5% for ferulic acid, p-hydroxybenzoic acid, vanillic acid, cinnamic acid, and benzoic acid, respectively. After 84 h of incubation, ferulic acid and p-coumaric acid could not be detected in the strain P5-inoculated culture, and only 3.1% p-hydroxybenzoic acid, 6.5% vanillic acid, and 4.7% benzoic acid were left in the strain P5-inoculated culture. After 96 h of incubation, all phenolic acids added in the strain P5-inoculated culture were completely degraded, and the dry weight of strain P5 mycelia increased to 0.89 g dry weight per liter culture.

In the greenhouse pot experiment, the highest disease incidence (56.3%) occurred in the CK treatment (Figure 7). The application of strains B2 and P5 significantly decreased the disease incidence by 48.1 and 22.2%, respectively, compared to the CK treatment. However, the combined treatment B2 + P5 showed the best control efficacy of Fusarium wilt by 63.0% (Figure 7).

The results showed that application of strain B2 or P5 alone was effective for growth promotion in cucumber plants (Table 3). Additionally, the combined treatment B2 + P5 showed significantly stronger growth promotion effect compared to B2 and P5 treatments (Table 3). Plant dry weight, plant height, root length, and root dry weight with the B2 + P5 treatment increased by 64.6, 26.4, 37.6, and 53.8% compared to the control, respectively.

A real-time PCR assay was used to determine the population of FOC in rhizosphere soil at the end of the experiments. The results indicated that the application of strain B2 or P5 alone significantly suppressed the population of FOC in rhizosphere soils, which was reduced by 74.0 and 30.5% compared to the CK treatment, respectively (Figure 8). The B2 + P5 treatment showed the strongest inhibitory effect on FOC population, which was only 1.62 × 10⁴ copies g^–1 soil in the B2 + P5 treatment, corresponding to 83.2% reduction compared to control treatment (Figure 8).

All inoculation treatments reduced the contents of phenolic acids in the soil (Table 4). Compared to the CK treatment, the contents of total phenolic acids were significantly (p < 0.05) decreased by 18.9, 35.9, and 63.2% in B2, P5, and B2 + P5 treatments, respectively (Table 4).

Correlation coefficients were calculated between the disease incidence and abundance of FOC and content of phenolic acids at 60 days after transplanting. Disease incidence was significantly positively correlated with FOC and contents of p-hydroxybenzoic acid, vanillic acid, ferulic acid, p-coumaric acid, benzoic acid, and total phenolic acids (p < 0.05; Figure 9).