Nests of O. formosanus were collected in July 2017 in Jiangyin City, Jiangsu Province, China. The termite was authenticated by Professor Jianguo Wang (Department of Plant Protection, College of Agriculture, Jiangxi Agricultural University). More than three termite nests were grinded in mortar, followed by the treatment with an equal volume of 70% (v/v) ethanol for 4 h at room temperature to kill vegetative cells. Subsequently, the material was washed three times with sterile water. The diluted sample was coated onto the Luria-Bertani (LB) culture medium (NaCl 10 g, peptone 10 g, yeast extract 5 g, and agar 15 g, in 1 L of distilled water), which was supplemented with 0.1% sodium taurocholate in order to promote spore germination (Browne et al., 2016). All mediums were cultured at 37°C for 24 h. After 2 days, a total of 30 germinal microorganisms were conserved on slant LB medium and stored at 4°C.

All the bacteria isolates were initially screened to assess their antifungal activity against F. oxysporum f. sp. cucumerinum (FOC) using a dual culture method on potato dextrose agar (PDA) plates (Ribeiro et al., 2021). A single 6 mm plug of the pathogen from 4 days of growth was inoculated in the center of the PDA medium. The isolates with 1 μl bacterial suspension (10⁸ cfu/ml) were spot inoculated at three sites equidistant from the center of Petri plates. PDA media inoculated with the pathogen alone were used as control. All the plates were cultured continuously at 28°C. After 4 days, the diameter of the pathogen colony (cm) was recorded. The inhibition percentage (I) was calculated using the following formula (Nasr et al., 2019):

I (%) = [(R – r)/R] × 100

where r and R are the radial growth of fungal pathogens in dual plate culture and control plates, respectively.

The antifungal spectrum of the selected strain YC-9 against the following plant pathogens was detected by the dual culture method described above: F. oxysporum f. sp. vasinfectum, Alternaria solani, Colletotrichum graminicola, Curvularia lunata, Corynespora cassiicola, F. oxysporum f. sp. mornordicae, Botrytis cinerea, and Fusarium graminearum. All plant fungal pathogens were obtained from the Department of Microbiology, School of Life Sciences, Anhui Agricultural University, Hefei, China.

The antagonistic isolates were identified using 16S rRNA gene sequences and phylogenetic analysis (Gorai et al., 2021). Total DNA was extracted using an Aidlab DNA extraction kit (Aidlab Biotechnologies Co., Ltd., Beijing, China). Primers 27F (5′-AGAGTTTGATCATGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used as amplification primers. Polymerase chain reaction amplification (PCR) was utilized on the basis of the following steps: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 90 s, and a final extension at 72°C for 10 min, with 30 cycles in total. DNA sample was sequenced by General Biol (Anhui) Co., Ltd. Additionally, 16S rRNA gene sequence-based phylogenetic analysis was performed by the Maximum Likelihood method with the MEGA 6.0 software. A bootstrap analysis with 1,000 replicates was performed to calculate node support.

After 15 days of cultivation, the cucumber seedlings were inoculated with different treatments, including sterile water (A), YC-9 suspension (B), FOC conidia suspension (C), and challenge inoculation with FOC after 48 h of YC-9 suspension inoculation (D). The experiment was set up for three replicates. An amount of 0.2 g of root samples was collected on the 2nd, 4th, 6th, 8th, and 10th days after inoculation and homogenized with liquid nitrogen. The homogenized tissues were rinsed with 1 ml of ddH₂O (25 mM borate buffer for the PAL assay) at 4°C. The tissue extracts were centrifuged at 5,000 × g for 5 min at 4°C. The supernatant was stored at –80°C for the enzymatic activity assays.

The detection of encoding genes of known antibiotics was determined by the method described by Gorai et al. (2021) with less modification. A PCR reaction was carried out in the mixture, which contained 1 μl (50 ng) of DNA template of YC-9, 1 μl of each primer (which was listed in Table 1), 12.5 μl of Premix Taq (LA Taq Version 2.0), and 9.5 μl of ddH₂O. PCR amplification was utilized on the basis of the following steps: pre-denaturation at 94°C for 10 min, denaturation at 94°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 2 min, and a final extension at 72°C for 10 min, with 35 cycles in total. The amplified product was subjected to electrophoresis with 1% agarose gel. The results were observed and recorded by a gel imager.

The fermentation of strain YC-9 was conducted according to the methods described in detail previously (Liu and Sun, 2021). The single colony of B. siamensis YC-9 strain was inoculated into a 250-ml flask containing 100 ml of LB medium and cultured at 180 r/min for 24 h at 28°C. Then, 5 ml of this seed culture was inoculated into a 1-L flask containing 500 ml of LB medium (× 39) and cultured at 180 r/min for 3 days at 28°C. The culture broth (19.5 L) was filtered by gauze to afford the supernatant. The supernatant was extracted with EtOAc (3 × 20 L) at room temperature. The EtOAc phase was evaporated in vacuo to afford a crude extract and then subjected to silica gel column elution with a stepwise gradient of CH₂Cl₂/MeOH (100:0–100:8, v/v) to give five fractions (Fr1-Fr5). Fr1 (CH₂Cl₂/MeOH, 100:0, v/v) was further fractionated on a silica gel column, eluting with a stepwise gradient of petroleum ether (PE)/ethyl acetate (EA) (100:0–100:10, v/v) to give compound 1 (9.5 mg). Fr3 (CH₂Cl₂/MeOH, 100:2, v/v) was repeatedly chromatographed over a silica gel column to give compound 2 (22.7 mg). Fr5 (CH₂Cl₂/MeOH, 100:8, v/v) was repeatedly chromatographed over a silica gel column to give compound 3 (46.8 mg). The remaining part of Fr5 was loaded onto a Sephadex LH-20 column (100% MeOH) to yield compounds 4 (38.4 mg) and 5 (6.1 mg). Due to the limitation of separation means, no monomer compound was isolated and purified from Fr2 and Fr4.

Structural identifications of the metabolites were made on the basis of the spectroscopic data and mass spectrometry. Nuclear magnetic resonance (NMR) spectra were recorded on an Agilent II DD2 instrument operating at 600 MHz for ¹H and 150 MHz for ¹³C, while the DEPT and 2D spectra (COSY, HMQC, and HMBC) were obtained using the standard Agilent software. Chemical shifts were given in parts per million (δ) downfield from the TMS internal standard. The electrospray ionization mass spectrometry (ESI–MS) spectra were determined on a micrOTOF II mass spectrometer (Mariner Mass 5304, United States).

The inhibitory activities of the secondary metabolites of strain YC-9 against spore germination of FOC were assessed as reported previously with slight changes (Zhang et al., 2013). FOC spores were obtained from PDA plates of 5-day-old cultures. All secondary metabolites were made to obtain solutions with serial concentrations in aqueous solution (1% acetone). A tested compound solution (60 μl) was added to a spore suspension (60 μl, 10⁵ spores/ml). Then, aliquots of 40 μl of spore suspension from each were placed on separate glasses in triplicate. Cycloheximide (Hefei Bomei Biotechnology Co., Ltd.) was used as the positive control. The negative control was treated with the above aqueous solution alone. Slides containing spores were incubated in a moisture chamber at 28°C for 6 h, after which approximately 100 spores were examined under a light microscope to determine the percentage of germinated spores. The percentage of spore germination inhibition was calculated from mean values as follows:

Inhibition (%) = (A – B)/A × 100

where A and B are the percentages of germinated spores in the control and the sample, respectively.

The contribution and significance of the treatments were determined using a one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test or Student’s t-test to determine significant differences between treatment means (p < 0.05) with the SPSS 20.0 software. All experiments were performed with at least three independent replicates, unless otherwise stated.

In total, 30 bacterial strains were isolated from the nest of the termite, O. formosanus, seven of which exhibited strong antifungal activity against FOC with the inhibition rates ranging from 45.7 to 72.5% in the dual culture (Figure 1). Among them, the strain YC-9 showed the strongest antifungal activity against FOC, with an inhibition rate of 72.5% (Supplementary Figure 1). Therefore, the strain YC-9 was selected as the objective strain in this study to further investigate its biocontrol effect.

The inhibitory activities of the strain YC-9 against eight selected pathogenic fungi are presented in Figure 2 and Supplementary Figure 2. The results showed that the strain YC-9 had the most potent inhibitory effect on all tested fungi except for F. graminearum. The strain YC-9 was found to have broad spectrum antifungal activity and could be applied to biological control of other crop diseases.

A 1,431 base pair (bp) strain of the 16S rRNA gene was amplified and sequenced. The sequence was then queried against the EzBioCloud database. Relevant type strains were selected to construct a phylogenetic tree using the maximum-likelihood method (Kumar et al., 2021). The phylogenetic analysis revealed that the strain YC-9 and B. siamensis clustered on the same branch, with 99.86% sequence similarity for the 16S rRNA gene sequence (Figure 3), indicating the strain YC-9 as B. siamensis.

In vivo challenge experiment was performed to check the ability of B. siamensis YC-9 to reduce the disease severity. After 20 days of treatment with conidial suspension of FOC (10⁸ cfu/ml), 32.8% disease severity was observed for control. However, only 8.8% of disease severity was recorded due to the application of B. siamensis YC-9 even after pathogenic treatment (Supplementary Figures 3, s4). The calculated control efficacy was 73.2%. This observation strongly suggested that the B. siamensis YC-9 had significant suppression activity against FOC even after treatment with the pathogenic conidia in high concentration.

The effects of inoculation with B. siamensis YC-9 on cucumber growth characteristics are presented in Table 2. The results showed that B. siamensis YC-9 increased the fresh weight by 42.6%, dry weight by 53.0%, plant height by 20.8%, and root length by 19.3%. Thus, the application of B. siamensis YC-9 could promote cucumber growth in comparison with those grown without the experimental strain.

The resistance-related enzymes (POD, PPO, and PAL) activities in the roots of cucumber seedlings were measured at different times after pathogen inoculation (Figure 4). The results showed that the POD activity of cucumber treated with B. siamensis YC-9 was significantly higher than other treatments after pathogen inoculation and increased to the top level of 2,401 U/g⋅Fw⋅h on the 4th day, while the group A (CK), group B (only B. siamensis YC-9 was inoculated), and group C (only FOC was inoculated) were only 818, 1,244, and 1,024 U/g⋅Fw⋅h, respectively. The activities of PPO and PAL after pathogen inoculation were the overall upward trend, and their activities were maximum on the 10th day with 380 and 18.6 U/g⋅Fw⋅h, respectively. The results showed that B. siamensis YC-9 treatment could significantly increase the activities of PAL, PPO, and POD for some period of time after treatment in contrast to other treatments.

The presence of genes for biosynthesis of antibiotics such as 2,4-diacetyl phloroglucinol (DAPG), pyrrolnitrin (PRN), bacillomycin, fengycin, iturin, and surfactin in B. siamensis YC-9 was confirmed by PCR analysis with gene-specific primers. The primer groups and PCR amplification conditions were listed in Table 1. The primer set SRFAF/R amplified a 1,300-bp fragment from the srfAA gene, involved in the biosynthesis of surfactin. The primer set FEND1F/R amplified a 964-bp fragment from the fenD gene, involved in the biosynthesis of fengycin. The primer set BACC1F/R amplified an 875-bp fragment from the bmyB gene, involved in the biosynthesis of bacillomycin. The primer set ItuC-F/R amplified a 465-bp fragment from ituC gene, involved in the biosynthesis of iturin. The primer sets for Phl2a and Phl2b amplified a 745-bp fragment from phlD gene, involved in the biosynthesis of DAPG. The primer sets for PrnCf/r amplified a 719-bp fragment from the prnC gene, involved in the biosynthesis of PRN. The results of electrophoresis showed that B. siamensis YC-9 contained genes for biosynthesis of bacillomycin, iturin, and surfactin (Supplementary Figure 5).

Chromatographic separation of the ethyl acetate extracts from the filtrate of B. siamensis YC-9 afforded five secondary metabolites (Figure 5), which were identified as hexadecanoic acid (1) (Aiyelaagbe et al., 2019), cyclo-(L-phenylalanylglycine) (2) (Zhuravleva et al., 2011), cyclo-(L-trans-Hyp-L-Leu) (3) (Xiang et al., 2020), C₁₅-surfactin (4) (Tang et al., 2007), and macrolactin A (5) (Schneider et al., 2007) by spectroscopic data (Supplementary Figures 6–10) analyses, and the comparison of their derivative data in the literature.

The isolated secondary metabolites from the culture of strain YC-9 were further evaluated for their antifungal activities by using the spore germination inhibition assay. Figure 6 showed C₁₅-surfactin (4) had significantly antifungal activity against spore germination of FOC when compared to cycloheximide co-assayed as a positive reference and had higher antifungal activity with the increasing concentration. The calculated IC₅₀ value of antifungal activity for C₁₅-surfactin (4) was 5.1 μg/ml, which was comparable to that of the positive control, cycloheximide (IC₅₀ value of 2.6 μg/ml). However, the other secondary metabolites were not active against spore germination (IC₅₀ > 50 μg/ml).