Bacillus subtilis B579 Controls Cucumber Fusarium Wilt by Improving Rhizosphere Microbial Community

Abstract

With continuous improvements in people's environmental awareness, biological control agents have garnered considerable attention owing to their advantageous impacts on improving soil fertility and alleviating plant diseases. Bacillus subtilis (B. subtilis) B579, isolated from the rhizosphere soil of cucumber, has effectively suppressed the growth of pathogenic Fusarium oxysporum. Our study investigates the effects of B. subtilis B579 on the properties of the rhizosphere soil (its physicochemical properties and enzymatic activities) and microbial community of cucumber under Fusarium oxysporum infection. An amplicon sequencing analysis of the microorganisms in the rhizosphere soil was conducted, and the soil's properties were measured. The findings demonstrated that B. subtilis B579 exhibited 73.68% efficacy in controlling cucumber Fusarium wilt disease. B579 pretreatment substantially increased the bacterial and fungi diversity and improved the soil's physicochemical properties (pH level and OC, TN, TP, AK, and AP contents) and enzyme activities, especially those of urease and alkaline phosphatase, which exhibited significant increases of 77.22% and 64.77%, respectively, in comparison to those under the pathogen treatment. Furthermore, the utilization of B579 reduced the abundance of Fusarium while simultaneously increasing the abundance of beneficial groups, including the Bacillus, Paenibacillus, Sphingomonas, Pseudomonas, Microbacterium, Mortierella, and Trichoderma genera. The RDA showed that the abundance of Bacillus, Paenibacillus, Sphingomonas, and Mortierella in the rhizosphere showed positive correlations with most of the soil properties, whereas Fusarium abundance was negatively correlated with most of the soil's properties. This study provides novel insights into the disease suppression mechanisms of Bacillus subtilis B579, laying the theoretical foundation for its development as a biocontrol agent.

Keywords: B. subtilis B579; biological control; cucumber Fusarium wilt; rhizosphere microorganism; soil environment.

Figure 1

The growth status of the cucumber seedings (a) and the disease index (b) of Fusarium wilt under different treatments. Note: Values represent the mean ± SE (n = 3). CK (cucumbers, irrigated with distilled water (control)); B579 (cucumbers irrigated with the B579 suspension (2 × 10⁸ CFU/mL)); B579 + FOC (BF) (cucumbers irrigated with the B579 suspension for 3 days and subsequently with the FOC suspension (1 × 10⁸ conidia/mL)); FOC (cucumbers irrigated with the FOC suspension); values with the same letter do not differ significantly (p < 0.05) according to Tukey’s test.

Figure 2

Growth indices of cucumber seedings in different treatments. (a) Plant height; (b) stem diameter; (c) fresh weight; (d) dry weight. CK (cucumbers irrigated with distilled water (control)); B579 (cucumbers irrigated with the B579 suspension (2 × 10⁸ CFU/mL)); B579 + FOC (BF) (cucumbers irrigated with the B579 suspension for 3 days and subsequently with the FOC suspension (1 × 10⁸ conidia/mL)); FOC (cucumbers irrigated with the FOC suspension). Note: Values represent the mean ± SE (n = 3). Values with the same letter do not differ significantly according to Tukey’s test (p < 0.05).

Figure 3

Venn diagram of the number of bacterial (a) and fungal (b) ASVs in different samples. CK (cucumbers irrigated with distilled water (control)); B579 (cucumbers irrigated with the B579 suspension (2 × 10⁸ CFU/mL)); B579 + FOC (BF) (cucumbers irrigated with the B579 suspension for 3 days and subsequently with the FOC suspension (1 × 10⁸ conidia/mL)); FOC (cucumbers irrigated with the FOC suspension).

Figure 4

A rarefaction analysis for the soil samples collected from the four treatments. CK (cucumbers irrigated with distilled water (control)); B579 (cucumbers irrigated with the B579 suspension (2 × 10⁸ CFU/mL)); B579 + FOC (BF) (cucumbers irrigated with the B579 suspension for 3 days and subsequently with the FOC suspension (1 × 10⁸ conidia/mL)); FOC (cucumbers irrigated with the FOC suspension). (a) Bacteria; (b) fungi. The vertical axis shows the average number of observed features found after sampling the number of sequences shown on the horizontal axis.

Figure 5

A principal coordinate analysis (PcoA) of the (a) bacterial and (b) fungal community from the cucumber rhizosphere soil samples. CK (cucumbers irrigated with distilled water (control)); B579 (cucumbers irrigated with B579 suspension (2 × 10⁸ CFU/mL)); B579 + FOC (BF) (cucumbers irrigated with B579 suspension for 3 days and subsequently with FOC suspension (1 × 10⁸ conidia/mL)); FOC (cucumbers irrigated with FOC suspension).

Figure 6

The relative abundance of the dominant phyla in the cucumber rhizosphere soil samples: (a) bacteria; (b) fungi. CK (cucumbers irrigated with distilled water (control)); B579 (cucumbers irrigated with B579 suspension (2 × 10⁸ CFU/mL)); B579 + FOC (BF) (cucumbers irrigated with B579 suspension for 3 days and subsequently with FOC suspension (1 × 10⁸ conidia/mL)); FOC (cucumbers irrigated with FOC suspension).

Figure 7

A heatmap of the top 30 classified bacterial genera for the samples in different treatments. CK (cucumbers irrigated with distilled water (control)); B579 (cucumbers irrigated with B579 suspension (2 × 10⁸ CFU/mL)); B579 + FOC (BF) (cucumbers irrigated with B579 suspension for 3 days and subsequently with FOC suspension (1 × 10⁸ conidia/mL)); FOC (cucumbers irrigated with FOC suspension). Data were normalized between groups, with red representing the high relative abundance of a species and blue representing low abundance of that species.

Figure 8

A heatmap of the top 15 classified fungal genera for the samples in different treatments. CK (cucumbers irrigated with distilled water (control)); B579 (cucumbers irrigated with B579 suspension (2 × 10⁸ CFU/mL)); B579 + FOC (BF) (cucumbers irrigated with B579 suspension for 3 days and subsequently with FOC suspension (1 × 10⁸ conidia/mL)); FOC (cucumbers irrigated with FOC suspension).

Figure 9

The redundancy analysis (RDA) based on the major genera and the soil’s physicochemical properties in the rhizosphere soil of cucumber. (a) Bacteria; (b) fungi. CK (cucumbers irrigated with distilled water (control)); B579 (cucumbers irrigated with B579 suspension (2 × 10⁸ CFU/mL)); B579 + FOC (BF) (cucumbers irrigated with B579 suspension for 3 days and subsequently with FOC suspension (1 × 10⁸ conidia/mL)); FOC (cucumbers irrigated with FOC suspension). TP, total phosphorus; TN, total nitrogen; OC, organic carbon; AK, available potassium; AP, available phosphorus; OP, organic phosphorus; UE, urease; ALP, alkaline phosphatase; CAT, catalase; CHI, chitinase; β-GLU, β-glucosidase.