All fungal and bacterial strains used in this study were isolated and preserved by the Institute of Chinese Herbal Medicines, Hubei Academy of Agricultural Sciences. The main strains, B. velezensis LT1 and S. rolfsii LC1, were stored in the China Center for Type Culture Collection Center with the numbers of CCTCC M 2020023 and CCTCC M 20231389, respectively.

Rhizosphere soil samples were collected from the C. chinensis GAP planting base (108°35′E, 30°22′N) located in Lichuan City, Enshi Prefecture, Hubei Province, in October 2019. The planting base had a history of perennial southern blight occurrence. The rhizosphere soil, which is located approximately 5–10 cm below the ground part of the C. chinensis plant, was collected using a JC-802 root soil sampler (Qingdao Juchuang Times Environmental Protection Technology Co., LTD, Qingdao, China). The rhizome samples obtained from five locations in the rhizosphere soil were uniformly mixed to form one sample.

Bacteria in the rhizosphere soil of C. chinensis were isolated using the dilution coating method, as described by Li et al. (2023). The soil samples were prepared into suspensions with concentrations of approximately 10⁻¹–10⁻⁶, and 50 μL of the soil dilutions was uniformly coated on the surface of NA medium, R2A medium, and brain-heart infusion liquid medium. The NA medium contained 10 g of tryptone, 3 g of beef powder, 5 g of NaCl, and 15 g of agar per liter. The R2A medium contained 0.5 g of yeast extract powder, 0.5 g tryptone, 0.5 g of casein hydrolysate, 0.5 g of glucose, 0.5 g soluble starch, 0.3 g of KH₂PO₄, 0.024 g of MgSO₄, 0.3 g of CH₃COCOONa, and 15 g of agar per liter. The brain-heart infusion liquid medium contained 5 g of tryptone, g of glucose, 5 g of NaCl, 2.5 g of Na₂HPO₄, 4 g of beef brain infusion, 16 g of casein peptone, and 15 g of agar per liter. The Petri dishes were then sealed and incubated at a constant temperature of 28°C. Colonies with distinct differences in morphology, size, and color were selected for purification. The colonies were purified three to four times using the scribing plate method until single colonies were observed. Single colonies were transferred to a bead-side culture medium test tube and stored in the refrigerator at 4°C for future use. Frozen stocks of all the purified colonies were prepared using 25% glycerol and stored at −80°C.

Bacterial genomic DNA was extracted using a Soil FastDNA SPIN Kit (MP Biomedicals (Shanghai) Co., Ltd., Shanghai, China). After assessing the quality of the genomic DNA extraction using a microspectrophotometer (NanoDrop One C, Thermo Fisher Scientific, Madison, United States), 16S rDNA was amplified using the primers 24F (5′-GTTTGATCCTGGCTCAG-3′) and 1494R (5′-ACGGCTACCTTGTTACGACTT-3′). The PCR products were purified and recovered by Wuhan Tsingke Biology Co., Ltd. for sequencing. The sequencing results were compared with the BLAST® sequence analysis tool at the US National Center for Biotechnology Information (NCBI) for preliminary identification of the isolated strains.

The plate confrontation method (Yue et al., 2023) was used to screen for antagonistic strains against S. rolfsii in C. chinensis. The activated S. rolfsii LC1 was inoculated with a mycelial plug using a hole punch of 5 mm diameter and placed in the center of the PDA medium with the mycelial side facing down. The isolated and purified bacteria were inoculated 2.5 cm away from the mycelial plug and cultured at 28°C for 4–7 days. The control group was not inoculated with the isolated strains, and each experiment was repeated three times. The colony diameter was measured using the crisscross method, and the inhibition rate was calculated using the following formula:

The LT1 strain exhibited the strongest inhibitory effect on S. rolfsii LC1. Therefore, only the LT1 strain was subjected to morphological and molecular analyses. Morphological characteristics of the LT1 strain were observed using microscopy LSM 900 (Carl Zeiss Co. Ltd., Chengdu, China) and scanning electron microscopy (HITACHI Regulus 8,100) (Hitachi Production Co., LTD., Tokyo, Japan). Subsequently, the LT1 strain underwent Gram staining using the Gram Stain Kit HB8278 (Haibo Biotechnology Co. LTD., Qingdao, China). The gyrA gene was amplified using the primers gyrA-F (5’-CAGTCAGGAAATGCGTACGTCCTT-3′) and gyrA-R (5’-CAAGGTAATGCTCCAGGCATTGCT-3′). The PCR products were subjected to 1% (w/v) agarose gel electrophoresis, purified, and sequenced by Wuhan Aoke Biotechnology Co., Ltd. The sequencing results were compared using the BLAST® sequence analysis tool in NCBI¹ and nucleic acid homology comparison with the data in GenBank. When constructing a phylogenetic tree using 16S rDNA and gyrA genes, bacteria with high homology to Bacillus were selected as ingroups and other bacteria were selected as outgroups. Clustal X was used to compare the sequences, and MEGA5.0 software (Mega Technology Co. LTD., Suzhou, China) was used to analyze the phylogeny of all bacteria. The two genes were combined to construct a phylogenetic tree, and the bootstrap value was tested 1,000 times. Finally, the optimum growth temperature and pH for the LT1 strain were determined.

To obtain the fermentation liquor, 1 mL of the LT1 strain was inoculated into 1,000 mL of liquid LB and incubated for 48 h at 180 r/min at 28°C. The fermentation solution was centrifuged at 3000 r/min for 10 min at 4°C, and the upper fermentation solution was filtered using a bacterial filter (0.22 μm). The inhibition rate of the resulting fermentation liquor on the mycelial growth of S. rolfsii LC1 was determined using a solid plate method. The LT1 fermentation liquor was prepared in 20 mL PDA plates at proportions of 2, 6, and 10% (v/v). PDA plates without the fermentation filtrate were used as controls. The mycelial plug of activated S. rolfsii LC1 (φ = 5 mm) was inoculated in the center of the PDA plate and cultured at 28°C for 3 days. The diameter of each colony was measured using the criss-cross method with three replicates per treatment. The mycelial growth inhibition rate was calculated using the following formula:

Since the 10% fermentation liquor exhibited the best inhibitory effect against S. rolfsii LC1, it was selected to assess the effect of LT1 strain against other pathogens such as Rhizoctonia solani ZJSQK1 of Panax japonicus, Sclerotinia sclerotiorum BXHP1 of Pinellia ternata, Fusarium acuminatum MD1 of Ophiopogon japonicus, Fusarium oxysporum QK-4 of tobacco, Colletotrichum micotianae YCTJ1 of tobacco, Alternaria alternata YCCX1 of tobacco, and Botrytis cinerea ES8 of Paris polyphylla. All the aforementioned pathogens were obtained from the Institute of Chinese Herbal Medicines of the Hubei Academy of Agricultural Sciences and preserved.

First, the fermentation liquid and filtered liquid of the LT1 strain were directly sprayed onto potted C. chinensis, and then, healthy leaves were selected to inoculate the activated S. rolfsii LC1. Each Potted C. chinensis was inoculated with three leaves. Potted C. chinensis inoculated with LC1 was then sealed in a plastic bucket to maintain moisture, and the incidence of southern blight was observed. Untreated C. chinensis were inoculated with S. rolfsii LC1 as a control group, which were treated with tifuramide as a positive control group, and each experiment was repeated three times.

From 150 mL of LT1 strain’s fermentation broth, 20 mL of acetic acid was incorporated to attain a pH of 3.0. The fermentation filtrate was then sequentially extracted with equal volumes (1:1 v/v) of ethyl acetate, dichloromethane, methanol, and acetone. The extracts were clarified by passing them through a 0.22 μm filter to remove any bacterial residues. Subsequently, these filtered extracts were incorporated into a PDA medium at a 1:10 (v/v) ratio to prepare agar plates. An actively growing mycelial plug of S. rolfsii LC1 (φ = 5 mm) was centrally inoculated onto each PDA plate. The plates were then incubated at 28°C for 3 days. As a control, an equal volume of each solvent was added to separate PDA plates. The experiment was conducted in triplicate for statistical robustness. The diameter of each treated colony was determined by the cross-cross method. The mycelial growth inhibition rate was calculated using the following formula:

The fermentation filtrate was extracted with a 1:1 (v:v) acetone solution. In total, 1 μL of the acetone extract was placed on a target containing the same amount of mother liquor (CHCA) in the mass spectrometer and air-dried naturally. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (Waters Corporation, Milford, MA, United States) was used for mass spectrometry analysis by a Crylas FTS355-Q laser (CryLaS, Berlin, Germany) source (355 nm) as the ion analytical ionization source, with a frequency of 1,000 HZ, laser energy of 250, and sample test range of 0–2000 m/z.

The fermentation filtrate of LT1 was mixed with PDA medium at a ratio of 1:9 (v/v) to prepare a plate. An activated S. rolfsii LC1 mycelial plug (φ = 5 mm) was inoculated into the center of the PDA plate and cultured at 28°C for 3 days. The mycelium was excised and rapidly submerged into glutaraldehyde solution for electron microscopy fixation for 2 hours, then washed three times with 0.1M phosphate buffer (PB) at pH 7.4. The mycelial morphology was observed using a scanning electron microscope (HITACHI Regulus 8,100) (Hitachi Production Co., LTD., Tokyo, Japan) and a transmission electron microscope (HITACHI HT7800) (Hitachi Production Co., LTD., Tokyo, Japan).

The data were analyzed using analysis of variance and Duncan’s multiple comparison tests with SPSS 22.0 (International Business Machines Corporation, New York, United States). The mean ± standard error (SEM) was used to express the data. Significance levels were indicated as ^* p < 0.05 and ^** p < 0.01 for two levels of significant differences. GraphPad Prism software (version 6.0, GraphPad Software, Calif., United States) was used for data visualization.

A total of 46 bacterial strains with different morphologies were obtained by coating the diluted soil suspension onto R2A, BHI, and NA media to obtain single colonies. The 46 strains were preliminarily identified by 16S rDNA that belong to 4 phyla, 9 classes, 10 orders, 17 families, 24 genera, and 32 species (Supplementary Table S1). The sequencing results of 16S rDNA for all strains are shown in Supplementary Data S1. The inhibitory effect of LT1 strain on the mycelial growth of S. rolfsii LC1 was measured using the face-off culture method. Many strains, including LT1, LT2, LT4, and SLT26, could significantly inhibit the growth of S. rolfsii LC1. Among these, the LT1 strain showed the best inhibitory effect (78.41%) (Figures 1A,B).

Morphological and molecular biology methods were employed to identify the specific species of the LT1 strain with the best antagonistic effect. The single colony of LT1 on the NA medium was round, rough, and white (Figure 1C). The purple Gram staining indicated that the LT1 strain was a gram-positive bacterium (Figure 1D). Scanning electron microscopy showed that the bacteria were rod-shaped with obtuse ends and smooth cell walls, ranging in size from 0.3 μm x 0.39 μm to 0.75 μm x 1.53 μm (Figure 1E). The 16S rDNA sequence of the LT1 strain with gene number OL677384 was found to be 99.8% similar to that of B. velezensis Blz02 (number: MN961683) from NCBI. The gyrA sequence of the LT1 strain with gene number OL692738 was 100% similar to that of B. velezensis Blz02 (number: MT395302) from NCBI. Based on the 16S rDNA sequence and gyrA sequence, a joint evolutionary tree was constructed, and the LT1 strain was clustered with the B. velezensis BY6, M75, and Hx05 strains. It could be effectively distinguished from other bacteria, such as B. amyloliquefaciens and B. subtilis (Figure 1F). Based on the morphological characteristics and molecular sequences, the LT1 strain was identified as B. velezensis LT1. The optimum pH for the growth of B. velezensis LT1 was 7, and it could not grow when the pH was lower than 3 or greater than 11 (Figure 1G). The optimum growth temperature was between 25 and 30°C, and B. velezensis LT1 could grow at temperatures ranging from 10 to 45°C (Figure 1H).

To assess the biocontrol potential of B. velezensis LT1 against southern blight of C. chinensis, we measured the inhibitory effect of the fermentation liquor without bacterial cells of B. velezensis LT1 on S. rolfsii LC1 in vitro and its control effect on potted C. chinensis. Three concentrations of B. velezensis LT1 fermentation filtrate showed inhibitory effects on the growth of S. rolfsii, with the inhibitory effects significantly varying among the concentrations. The inhibitory effect increased gradually as the fermentation filtrate concentration increased, reaching 90.43% when the fermentation filtrate concentration reached 10% (Figure 2A). We used a 10% fermentation filtrate to measure the inhibitory effects of B. velezensis LT1 on other pathogens. The results showed that B. velezensis LT1 had strong inhibitory effects on R. solani ZJSQK1 of P. japonicus, S. sclerotiorum BXHP1of P. ternata, F. acuminatum MD1 of O. japonicus, C. micotianae YCTJ1 of tobacco, A. alternata YCCX1 of tobacco, and B. cinerea ES8 of P. polyphylla (Figure 2B). These results demonstrate the high potential of B. velezensis LT1 for plant disease control. To further evaluate the control efficacy of B. velezensis LT1 against the southern blight of C. chinensis, we conducted pot experiments using different concentrations of fermentation liquid and filtrate. The control efficacy of 10% fermentation liquid and fermentation filtrate of B. velezensis LT1 against southern blight on potted C. chinensis reached 65%, whereas that of 1% fermentation liquid and fermentation filtrate of B. velezensis LT1 was approximately 40% (Figure 2C). The control efficacy of the 10% fermentation liquid was similar to that of the chemical fungicide tifuramide (Figure 2C). Notably, the same concentrations of fermentation liquid and filtrate without bacterial cells of B. velezensis LT1 showed equal control efficacy.

The fermentation liquid without bacterial cells of B. velezensis LT1 was found to significantly inhibit the growth of S. rolfsii LC1. This led us to suspect that B. velezensis LT1 may have produced substances in the fermentation liquid that inhibit fungal growth. To identify the specific components of these substances, B. velezensis LT1 fermentation liquid was extracted using different solvents. Among the five extractants tested, the fermentation liquid extracted with acetone showed the best inhibitory effect with a mycelial inhibition rate of S. rolfsii LC1 of 99%, followed by petroleum ether extraction with an inhibition rate of 61.28%. The inhibition rates of the extraction products using the other three extractants were lower than 45% (Figure 2D). Therefore, the acetone extract of B. velezensis LT1 fermentation liquid was subjected to MALDI-TOF-MS (m/z 0–2000) analysis, and the resulting lipopeptide secondary metabolites (m/z 0–2000) are shown in Supplementary Data S2. B. velezensis LT1 was found to produce multiple antimicrobial compounds, including bacillomycin D, bacillomycin LC, iturin A, fengycin A, and surfactin (Table 1). Many of these antimicrobial compounds have been reported to inhibit the growth of fungal pathogens. These results suggest that the production of rich lipopeptide antimicrobial compounds by B. velezensis LT1 may be the primary mechanism by which it inhibits S. rolfsii LC1.

To further explore the effect of B. velezensis LT1 on S. rolfsii LC1, we observed the mycelial morphology of S. rolfsii LC1 under B. velezensis LT1 stress using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM observations showed that normal mycelia were arranged in an orderly manner with a smooth surface, uniform thickness, good extensibility, and a complete cell wall (Figure 3A). However, under the stress of B. velezensis LT1, the mycelia of S. rolfsii LC1 were folded with partial mycelial rupture, cytoplasmic leakage, and mycelial fragmentation (Figure 3B). Transmission electron microscopy showed that the control mycelia grew normally, with uniform and complete cell walls, clear boundaries, light cytoplasm, uniform distribution, clear nuclei, abundant and complete organelles evenly distributed in the cytoplasm, and a clear structure (Figure 3C). After treatment with B. velezensis LT1, the mycelial cell walls were significantly thickened, the cytoplasm was concentrated and dark in color, and a large number of white particles appeared. The nuclear structure was unclear, and no obvious nuclear membrane boundary was observed. Additionally, the number of mitochondria was significantly reduced (Figure 3D). These observations suggest that B. velezensis LT1 may have a destructive effect on the mycelial structure of S. rolfsii LC1.