Mechanism of a novel Bacillus subtilis JNF2 in suppressing Fusarium oxysporum f. sp. cucumerium and enhancing cucumber growth

Abstract

Cucumber Fusarium wilt caused by Fusarium oxysporum f. sp. cucumerium (FOC), is a prevalent soil-borne disease. In this study, Bacillus subtilis JNF2, isolated from the high incidence area of cucumber Fusarium wilt in Luoyang, demonstrated significant inhibitory effects on FOC and promoted cucumber seedling growth. The biocontrol mechanism of strain JNF2 were elucidated through morphological observation, physiological and biochemical experiments, and whole genome sequence analysis. Pot experiments revealed an 81.33 ± 0.21% control efficacy against Fusarium wilt, surpassing the 64.10 ± 0.06% efficacy of hymexazol. Seedlings inoculated with JNF2 exhibited enhanced stem thickness and leaf area compared to control and hymexazol-treated plants. Physiological tests confirmed JNF2's production of indole-3-acetic acid (IAA), siderophores, and hydrolytic enzymes, such as β-1,3-glucanase, amylase, and protease, which inhibited FOC growth and promoted plant development. Genome analysis identified genes encoding antimicrobial peptides and hydrolases, as well as a novel glycocin synthetic gene cluster. These findings underscore B. subtilis JNF2's potential as a biocontrol agent for sustainable cucumber cultivation.

Keywords: Bacillus subtilis; Fusarium oxysporum f. sp. cucumerinum; biocontrol bacteria; growth promotion; whole genome sequence.

Figure 1

Biocontrol effect of strain JNF2. (A) Foc-FJH36 colony morphology without JNF2 strain on PDA plate; (B) Colony morphology of Foc-FJH36 containing JNF2 strain on PDA plate; (C) The morphological development of Foc-FJH36 was normal; (D) JNF2 strain caused the contraction of Foc-FJH36 mycelium; (E) JNF2 strain caused Foc-FJH36 to shrink and wilt; (F–J) From left to right, the results of indole-3-acetic acid (IAA), β-1,3-glucanase activity, amylase activity, protease activity and siderophore of JNF2 strain were detected.

Figure 2

The control effect of strain JNF2. (A) The control effect of B. subtilis JNF2; (B) The control effect of Hymexazol; (C) Cucumber seedlings inoculated with FOC.

Figure 3

Growth-promoting effect of strain JNF2. (A–C) Cucumber seedlings were cultured for 20 days; (D) Cucumber seedlings were cultured for 40 days.

Figure 4

The growth-promoting effect of strain JNF2 on cucumber plants at 20 and 40 days. (A) Plant height, root length and leaf area of cucumber seedlings at 20 and 40 days post-inoculation (dpi); (B) The stem diameter of cucumber seedlings at 20 and 40 dpi; (C) The chlorophyll content of cucumber seedlings at 20 and 40 dpi; (D) The strong seedling index of cucumber at 20 and 40 dpi; (E) The root and shoot dry weight of cucumber seedlings at 20 and 40 dpi; (F) Root and shoot fresh weight of cucumber seedlings at 20 and 40 dpi after inoculation.

Figure 5

Basic information of whole genome sequencing, assembly, functional annotation and identification of strain JNF2. (A) Read the coverage depth distribution map; (B) Statistical analysis of common and unique annotations of strain JNF2 basic database; (C) Genome circle map of strain JNF2; (D) Species identification of strain JNF2.

Figure 6

Annotation results of strain JNF2 basic function database. (A) Top10 species distribution map of NR library; (B) SwissProt library Top10 species distribution map; (C) GO annotation results bubble diagram of strain JNF2; (D) The second level of KEGG annotation results of strain JNF2; (E) COG functional classification map of strain JNF2; (F) TCDB classification statistical chart of strain JNF2.

Figure 7

CAZymes analysis of strain JNF2 genome and genome comparison between strain JNF2 and other three strains. (A) Carbohydrate enzyme statistics of B. subtilis JNF2 genome; (B) Functional characterization of glycoside hydrolase family of strain JNF2 based on CAZymes; (C) The collinearity analysis circle diagram of JNF2 and the other three strains. The innermost circle was the comparison of multi-strain JNF2 with the other three strains, followed by the n ratio, GC skewness, gene density and GC ratio of the genomes of the four strains; (D) Venn diagram showed the number of unique and common gene clusters between JNF2 and the other three strains; (E) Schematic diagram of homologous gene clusters in different species.

Figure 8

Prediction of four biocontrol mechanisms of Bacillus subtilis JNF2.