Background: Bacillus subtilis strain NCD-2 is an excellent biocontrol agent against plant soil-borne diseases and shows broad-spectrum antifungal activities. This study aimed to explore some secondary metabolite biosynthetic gene clusters and related antimicrobial compounds in strain NCD-2. An integrative approach combining genome mining and structural identification technologies using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (UHPLC-MS/MS), was adopted to interpret the chemical origins of metabolites with significant biological activities. Results: Genome mining revealed nine gene clusters encoding secondary metabolites with predicted functions, including fengycin, surfactin, bacillaene, subtilosin, bacillibactin, bacilysin and three unknown products. Fengycin, surfactin, bacillaene and bacillibactin were successfully detected from the fermentation broth of strain NCD-2 by UHPLC-QTOF-MS/MS. The biosynthetic gene clusters of bacillaene, subtilosin, bacillibactin, and bacilysin showed 100% amino acid sequence identities with those in B. velezensis strain FZB42, whereas the identities of the surfactin and fengycin gene clusters were only 83 and 92%, respectively. Further comparison revealed that strain NCD-2 had lost the fenC and fenD genes in the fengycin biosynthetic operon. The biosynthetic enzyme-related gene srfAB for surfactin was divided into two parts. Bioinformatics analysis suggested that FenE in strain NCD-2 had a similar function to FenE and FenC in strain FZB42, and that FenA in strain NCD-2 had a similar function to FenA and FenD in strain FZB42. Five different kinds of fengycins, with 26 homologs, and surfactin, with 4 homologs, were detected from strain NCD-2. To the best of our knowledge, this is the first report of a non-typical gene cluster related to fengycin synthesis. Conclusions: Our study revealed a number of gene clusters encoding antimicrobial compounds in the genome of strain NCD-2, including a fengycin synthetic gene cluster that might be unique by using genome mining and UHPLC–QTOF–MS/MS. The production of fengycin, surfactin, bacillaene and bacillibactin might explain the biological activities of strain NCD-2.

Bacillus spp. is one kind of the important representative biocontrol agents against plant diseases and promoting plant growth. In this study, the whole genomic sequence of bacterial strain HMB26553 was obtained. A phylogenetic tree based on the genome and ANI (average nucleotide identity), as well as dDDH (digital DNA–DNA hybridization), was constructed, and strain HMB26553 was identified as Bacillus velezensis. Fourteen biosynthetic gene clusters responsible for secondary metabolite were predicted via anti-SMASH, and six secondary metabolites were identified by UHPLC-QTOF-MS/MS (ultra-high-performance liquid chromatography coupled to quadrupole-time-of-flight tandem mass spectrometry). When the phytopathogen Rhizoctonia solani was treated with B. velezensis HMB26553, the mycelial structure changed, ROS (reactive oxygen species) accumulated, and the mitochondrial membrane potential decreased. Characteristics of strain HMB26553 were predicted and confirmed by genomic information and experiments, such as producing IAA, siderophore, extracellular enzymes and biofilm, as well as moving and promoting cotton growth. All these results suggested the mechanisms by which B. velezensis HMB26553 inhibits pathogen growth and promotes cotton growth, which likely provided the potential biocontrol agent to control cotton Rhizoctonia damping-off.

Bacillus subtilis NCD-2 demonstrates exceptional biocontrol potential against cotton Verticillium wilt. While previous studies have established its direct antifungal activity (e.g., inhibiting Verticillium dahliae mycelial growth and spore germination), our work reveals a novel mechanism: NCD-2 primes systemic resistance in cotton by activating plant immune-signaling pathways. Firstly, transcriptional profiling uncovered that NCD-2 triggers a defense response in roots analogous to V. dahliae infection, allowing cotton to maintain a more balanced state when confronted with pathogen attacks. Meanwhile, the mutant strains ∆fen and ∆srf—defective in lipopeptide synthesis—also improved cotton resistance to Verticillium wilt by activating partially identical defense pathways in cotton roots. Furthermore, the application of lipopeptide compounds derived from NCD-2, particularly surfactin and fengycin, could enhance host resistance to V. dahliae. Using an RT-qPCR approach, we found that numerous resistance-related genes were induced by these lipopeptide compounds. The up-regulation of SA/JA pathway markers (e.g., NPR1, ICS1, COI1, and LOX1) revealed NCD-2’s activation of plant immune signaling. Using virus-induced gene silencing (VIGS), we conclusively linked SA and JA signaling to NCD-2-induced defense priming. Silencing either pathway abolished resistance, highlighting their indispensable coordination. By bridging mechanistic insights and agricultural applicability, our work positions NCD-2 as a sustainable alternative to conventional fungicides, addressing both crop productivity and environmental health.

Bacillus subtilis strain NCD-2 is a promising biocontrol agent for soil-borne plant diseases and shows potential for promoting the growth of some crops. The purposes of this study were to analyze the colonization ability of strain NCD-2 in different crops and reveal the plant growth promotion mechanism of strain NCD-2 by rhizosphere microbiome analysis. qRT-PCR was used to determine the populations of strain NCD-2, and microbial communities’ structures were analyzed through amplicon sequencing after application of strain NCD-2. Results demonstrated that strain NCD-2 had a good growth promotion effect on tomato, eggplant and pepper, and it was the most abundant in eggplant rhizosphere soil. There were significantly differences in the types of beneficial microorganisms recruited for different crops after application of strain NCD-2. PICRUSt analysis showed that the relative abundances of functional genes for amino acid transport and metabolism, coenzyme transport and metabolism, lipid transport and metabolism, inorganic ion transport and metabolism, and defense mechanisms were enriched in the rhizospheres of pepper and eggplant more than in the rhizospheres of cotton, tomato and maize after application of strain NCD-2. In summary, the colonization ability of strain NCD-2 for five plants was different. There were differences in microbial communities’ structure in rhizosphere of different plants after application of strain NCD-2. Based on the results obtained in this study, it was concluded that the growth promoting ability of strain NCD-2 were correlated with its colonization quantity and the microbial species it recruited.