A. konjac: A. konjac (A. konjac K. Koch) was provided by Yunnan Jingtian Konjac Agriculture Co., Ltd.

Biocontrol strain: B. velezensis GZY0 was isolated and screened from the rhizosphere soil of A. konjac in Damogu Village, Luliang County, Qujing City, Yunnan Province (24.9361 N, 103.5809 E). The genome of this strain has been sequenced and uploaded to NCBI (SRP636943).

Pathogens: P. aroidearum, Botryosphaeria dothidea, F. oxysporum, F. solani, F. oxysporum f. sp. panax, and Colletotrichum gloeosporioides were provided by the Department of Plant Protection, Kunming University.

Culture media and reagents: LB solid medium (Haibo, China), LB liquid medium (Haibo, China), CAS solid medium (Haibo, China), Pikovskaya inorganic phosphorus medium (Nautiyal, 1999), and organic phosphorus solid medium were prepared as described previously (Nautiyal, 1999), with minor adjustments (10 g/L glucose, 2 g/L Phytin, 5 g/L MgCl₂·6H₂O, 0.25 g/L MgSO₄·7H2O, 0.2 g/L KCl, 0.1 g/L (NH₄)₂SO₄, and 15 g/L agar powder). Meanwhile, Salkowski reagent (Patten and Glick, 2002; Khan et al., 2019), the CAS detection solution (Louden et al., 2011), Ashby nitrogen-free medium (Abdallah et al., 2018), and MKB liquid medium were prepared as described by Wang et al. (2022), with minor adjustments (5.0 g of casamino acid, 15.0 mL of glycerol, 2.5 g of K₂HPO₄, and 0.2 g of MgSO₄·7H₂O; pH = 7.0).

Preparation of bacterial suspension: the target strain was cultured overnight on LB solid medium, transferred to sterile LB liquid medium, and incubated at 28 °C and 120 r/min for 48 h. The culture was centrifuged at 10,000 r/min for 5 min, and the cells were re-suspended in sterile water until the OD600 reached 0.3.

Preparation of cell-free supernatant (CFS): after overnight incubation on LB solid medium, the target strain was transferred to sterile LB liquid medium and incubated at 28 °C and 120 r/min for 48 h. The culture was centrifuged at 10,000 r/min for 5 min, and the supernatant was isolated. The CFS was obtained by filtering the aforementioned supernatant through a 0.22 μm sterile filter membrane. The sterility of the CFS was confirmed by plating 100 μL onto LB agar plates and observing no microbial growth after 72 h of incubation at 37 °C.

A total of 2 g of soil sample was added to 18 mL of sterile water containing glass beads, and the suspension was shaken for 20 min at 120 r/min and 28 °C. The suspension was serially diluted to 10-2, 10-3, and 10-4 in sterile water, and 100 μL of each dilutions were plated onto LB solid agar and incubated at 28 °C for 1–2 d. The colonies were picked up and steaked separately onto LB solid agar. The purified isolates were stored at 4 °C for antibacterial activity assays. The plate confrontation method (Yu et al., 2011) was used to determine the antibacterial activity of the purified isolates against P. aroidearum with minor modifications. Specifically, 150 μL of P. aroidearum suspension was added to 150 mL of sterilized LB agar medium (pre-cooled to 50 °C), the mixture was thoroughly mixed and poured into sterile plate, and cooled at room temperature until fully solidified. A single colony of potential antagonistic bacteria was spotted on the LB plate containing P. aroidearum, and incubated at 28 °C for 2 d. The antagonistic activities against P. aroidearum were evaluated by measuring the diameter of the inhibition zone (mm) around the isolated bacteria. The candidate strain GZY0 with the best antagonistic activity against P. aroidearum was selected.

Holes (5 mm in diameter) were punched in the center of the plate using a sterile perforator, and 10 μL of bioprophylaxis sterile fermentation solution was added to the holes to determine the diameter of the ring of inhibition (Ma et al., 2023).

Strain GZY0 was inoculated in LB liquid medium, incubated at 28 °C and 120 r/min overnight, and centrifuged at 4 °C and 8,000 r/min for 10 min. The cells were collected in centrifuge tubes after discarding the supernatant and washed thrice with PBS before freezing in liquid nitrogen for 1 h. The tubes were placed on dry ice and sent to Shanghai Major Biomedical Technology Co., Ltd. for whole-genome sequencing using the second-generation and third-generation sequencing method of Illumina and BacBio.

Based on ANI analysis, five closely related Bacillus spp. strains with fully sequenced genomes, including B. velezensis FZB42, B. velezensis SQR9, B. siamensis KCTC 13613, B. amyloliquefaciens DSM 7, and B. amyloliquefaciens NRRL B-14393, were selected to genomic comparison using the Shanghai Majorbio Bio-pharm Technology Co., Ltd. cloud platform.

To evaluate the effect of GZY0 cell suspension on soft rot disease caused by P. aroidearum, A. konjac tuber tissue inoculation was performed as described by Cui et al. (2021), with slight improvements. Healthy flowering konjac bulbs were taken and cut into 2 cm × 2 cm × 1.5 cm konjac bulb tissues, surface disinfected with 75% ethanol and sodium hypochlorite, and a 2.8 cm × 2.8 cm crisscross wound was made on the surface of the konjac block, inoculated with 20 μL of a 1:1 (v/v) mixture of P. aroidearum bacterial suspension (OD600 = 0.3) + GZY0 bacterial suspension, with 20 μL of Sterile water was used as blank control. Seven d later, the onset of the disease was observed and the disease index and control efficacy were counted (Hamaoka et al., 2021).

Grading standards are as follows. Level 0: konjac tuber is normal, no disease; Level 1: the disease area accounted for konjac tuber surface area of 1/8–1/4 (including 1/4); Level 2: the disease area accounted for konjac tuber surface area of 3/8–1/2 (including 1/2); Level 3: the disease area accounted for konjac tuber surface area of 5/8–3/4 (including 3/4); Level 4: the disease area accounted for konjac tuber surface area of the konjac tuber more than 7/8 (including 7/8).

Disease index = [∑(number of infections at each level × number of corresponding levels)]/(total number of investigations × number of the highest level) × 100%

Control efficacy = (control disease index – treatment disease index)/control disease index × 100%

To evaluate the effect of CFS of GZY0 on soft rot disease caused by P. aroidearum, a modified version of the method described by Li et al. (2019) was employed. A. konjac corm was cut in half, and its surface was perforated using a sterile perforator (diameter = 6 mm) and inoculated with 20 μL of a 1:1 (v/v) mixture of P. aroidearum bacterial suspension (OD600 = 0.3) + CFS of GZY0. The control treatment mixture was prepared using an equal amount of sterile LB medium instead of the GZY0 CFS. Sterile LB medium alone was used for the blank control. The incidence of soft rot was observed after 7 d, and the filter paper was kept moist with sterile water. The grading, disease index, and control efficacy were evaluated as described in Section 2.3.1.

For the nitrogen fixation test, 10 μL of the GZY0 bacterial suspension (OD600 = 0.3) was added to the center of an Ashby solid medium plate. The plate was inverted and incubated at 28–30 °C for 5 d to observe whether the bacterial colonies could grow normally (Wang et al., 2020; Wu et al., 2019).

For the phosphorus solubilization test, the GZY0 bacterial suspension (OD600 = 0.3) was inoculated onto a perforated (6 mm) organic phosphorus and inorganic phosphorus medium and incubated at 28 °C for 5 d. The presence of transparent rings was observed, and their diameter was measured.

For the siderophore assay, the GZY0 bacterial suspension (OD600 = 0.3) was inoculated onto CAS culture medium, and the presence of an orange halo was observed (Milagres et al., 1999). If such a halo was detected, quantitative analysis was carried out, as described previously (Machuca and Milagres, 2003).

To determine the IAA-producing ability of GZY0, the colorimetric Salkowski reagent method was used (Glickmann and Dessaux, 1995). If the test solution turned pink, quantitative analysis was performed. IAA standard solutions of 0 mg/L, 10 mg/L, 20 mg/L, 30 mg/L, 40 mg/L, and 50 mg/L were prepared, and their absorbance values were measured at 530 nm (OD530). The standard curve (y = 0.0069x + 0.0057, R2 = 0.9988) was plotted with the OD530 value along the vertical axis and the IAA concentration along the horizontal axis (Supplementary Figure S1). The IAA yield of strain GZY0 was calculated based on this standard curve.

The inhibitory effect of strain GZY0 against different pathogenic fungi was determined using the plate confrontation test. Preserved pathogenic strains were inoculated onto PDA plates for activation, while GZY0 was inoculated on LB solid medium. Strain GZY0 and pathogenic fungi were simultaneously inoculated on PDA plates, with the pathogenic fungi inoculated at the center of the plate. The diameter of the fungal plugs was 6 mm, and strain GZY0 was inoculated at an equal distance of 2.5 cm from the center of the plug using a toothpick, with three replicates per dish. The plates were incubated at 28 °C, and the fungistatic rate of strain GZY0 against the pathogens was determined based on fungal growth:

Fungistatic rate = [(control colony diameter – treatment colony diameter)/control colony diameter] × 100%.

Healthy A. konjac seedballs of uniform size were selected and planted in sterilized plastic pots with a base diameter of 7.6 cm, height of 9 cm, and inner diameter of 10 cm, with one plant in each pot. The planting substrate consisted of sterilized soil, organic soil, and vermiculite at a 1:2:2 ratio. There were three groups in total, with 15 pots per group, yielding a total of 45 pots. Cultivated in a greenhouse maintained at 28 °C and 75% relative humidity, the roots were irrigated with the GZY0 bacterial suspension (OD600 = 0.3) (50 mL per pot) once every 3 d for a total of three rounds. Control plants were treated with an equivalent amount of water instead of GZAY0. Three days after the irrigations were completed, wounds were induced on each A. konjac corm using a sterile syringe, and 50 mL P. aroidearum bacterial suspension (OD600 = 0.3) was added for root irrigation and inoculation. After inoculation, normal water and fertilizer management were administered. After 30 d of growth, the corm was removed, and the disease index and control efficacy were calculated as described in Section 2.5.

Grading standards are as follows. Level 0: no disease; Level 1: the onset of the area of the surface area of the konjac bulb 0%−20%; Level 2: the onset of the area of the surface area of the konjac bulb of 21%−40%; Level 3: the onset of the area of the surface area of the konjac bulb of 41%−60%; Level 4: the onset of the area of the surface area of the konjac bulb 61%−80%; Level 5: the onset of the area of the surface area of the konjac bulb of 81% and more than the konjac bulb. and above.

All the above experiments were repeated at least three times, and the results were expressed as the mean ± standard deviation (SD). All data were processed using Microsoft Excel 2021 and analyzed based on one-way analysis of variance (ANOVA) using IBM Statistics SPSS 27.0. Differences between groups were evaluated with Duncan's test, and a P-value of < 0.05 was considered statistically significant. The results of whole-genome sequencing were analyzed by the Major BioCloud platform.

A total of 59 strains were isolated and purified from the rhizospheric soil of A. konjac. One strain with significant inhibitory effects against the pathogen causing soft rot in A. konjac was selected after primary and secondary screening and named GZY0. Compared with the control (Figure 1Ab), both the cell suspension and CFS of GZY0 showed obvious inhibitory effects against P. aroidearum, producing zones of inhibition with diameters of 15.63 mm (Figure 1Aa) and 16.70 mm (Figure 1Ac), respectively.

Strain GZY0 exhibits milky-white, opaque, and smooth colonies on LB agar plates and rod-shaped cells with oval spores under microscopy. Furthermore, strain GZY0 was identified as a Gram-positive bacterium (Figure 1B). The 16S rRNA and gyrA sequences of GZY0 were subjected to BLAST alignment (NCBI), and their lengths were 1,423 and 935 bp, respectively. The similarity between the 16S rRNA sequences of strain GZY0 and B. velezensis HN2 (PP106361.1) was 99.86% (Figure 1C). Similarly, the similarity between the housekeeping gene gyrA sequences of strain GZY0 and the B. velezensis isolate LYi1 (ON221477.1) was 100% (Figure 1D). Accordingly, strain GZY0 was identified as a strain of B. velezensis.

GZY0 had the highest ANI value of 98.88% with B. velezensis FZB42 and the second highest ANI value of 97.87% with B. velezensis SQR9. The ANI values of GZY0 with the above two Bacillus species were higher than 96%, and the ANI values of GZY0 with the other Bacillus species were 65.79%−94.13%. This further confirmed that strain GZY0 belonged to B. velezensis (Figure 1E).

The A. konjac tuber tissue inoculation test revealed that the bacterial suspension and CFS of GZY0 exerted good in vitro antagonistic effects against soft rot in A. konjac. Compared to the control (Figures 2A, D), after 7 d of inoculation with P. aroidearum alone, the tuber tissue of A. konjac showed obvious morbidity and strong malodor (Figures 2B, E). However, after mixed inoculation with the GZY0 bacterial suspension and the CFS, tuber morbidity was significantly reduced (Figures 2C, F), and there was no bad odor; the control efficacy was 47.56% and 45.79%, respectively (Table 1).

Strain GZY0 could grow on nitrogen fixation medium (Figure 3A), which indicated that it could fix nitrogen. GZY0 also produced a transparent phosphorus halo on inorganic phosphorus and organic phosphorus media [11.68 mm (Figure 3B) and 7.50 mm (Figure 3C), respectively], which suggested that it had the ability to dissolve both inorganic and organic phosphorus.

GZY0 produced an orange-yellow halo with a diameter of 11.33 mm on CAS iron-loaded medium (Figure 3D), demonstrating its siderophore production capacity. Its A/Ar ratio was found to be 1.19 (Figures 3E, F). Additionally, the GZY0 suspension turned red following the addition of Salkowski's reagent (Figures 3G, H), which suggested that this GZY0 can produce IAA. The IAA yield of strain GZY0 was quantitatively determined to be 17.77 mg/L.

In plate confrontation tests involving different pathogens, GZY0 exhibited fungistatic activity against C. gloeosporioides, F. solani, B. dothidea, F. oxysporum f. sp. panax, and F. oxysporum, with fungistatic rates of 30–55% (Table 2). Notably, compared to the control group (Figures 3I–M), GZY0 exhibits varying inhibition rates against different pathogens (Figure 3i–m), with the strongest fungistatic activity against B. dothidea, with fungistatic rates up to 53.17% (Figure 3k). The weakest fungistatic activity was against F. solani (30.36%) (Figure 3j).

Using the unicycler assembly software, the GZY0 genome was assembled into a single circular chromosome with a total length of 4053804 bp and a sequencing depth of 349.84. The completeness score of 99.51% assessed by CheckM indicates that the assembly of GZY0 exhibits high integrity. Its G+C content was 46.53%, of which the A, T, G, and C contents were 1084228 bp, 1083494 bp, 943447 bp, and 942635 bp, respectively. A total of 3,934 coded genes were identified, with a total length of 3590091 bp, for one chromosome. Subsequently, tRNA and rRNA analyses showed that GZY0 contained 86 tRNAs and 27 rRNAs (Figure 4). A total of 130 genes were annotated through the CAZy database, and the number of genes enriched for glycoside hydrolases was 42, higher than that for other categories. Glycosyl transferases and carbohydrate esterases were linked to 41 and 32 genes, respectively (Supplementary Figure S2A).

In order to further analyze the genome of GZY0, sequencing data from GZY0 were aligned to the NR, COG, GO, SwissProt, KEGG, and Pfam databases. A total of 2,142 genes were annotated across the six databases, among which 3,928 genes were unique to the NR database, 3,168 genes to the COG database, 1,676 to the GO database, 3,602 to the SwissProt database, 2,898 to the KEGG database, and 3,458 to the Pfam database. Analysis via the COG database revealed that the genes were mainly involved in four groups of processes, including amino acid transport and metabolism, transcription, carbohydrate transport and metabolism, and general function only. Herein, amino acid transport and metabolism was linked to 311 genes; transcription, 300 genes; carbohydrate transport and metabolism, 287 genes; and general function only, 258 genes (Supplementary Figure S2B). Additionally, 807 genes were predicted in the PHI database using the Diamond software.

The GO database describes which biological, molecular functions, and cellular environments specific coding genes are involved in. The GO database contains three modules—Biological Process, Molecular Function, and Cellular Component. In this study, at the first level of classification, the highest number of genes from GZY0 were annotated to the Molecular Function module, followed by the Biological Process module. Among the secondary classifications, the highest number of genes were enriched for “membrane” in the Cellular Component module (345 genes). In the Biological Process module, the number of genes enriched for translation and phosphorylation (59 and 55 genes, respectively) was the highest. Finally, in the Molecular Function module, 228 and 188 genes were enriched for ATP binding and DNA binding, respectively. The results of GO analysis were important for subsequent genome function mining analysis (Supplementary Figure S2C). KEGG analysis showed that Metabolism was linked to the highest number of genes (i.e., 2,204) at the first level classification. Among these genes, 857 were involved in global and overview maps, accounting for the highest proportion of all secondary classifications. Carbohydrate metabolism was also enriched for a high number of genes (285 genes), while 229 genes were annotated to Amino Acid Metabolism and 216 genes were annotated to Metabolism of Cofactors and Vitamin. Hence, these three metabolic pathways likely played an important role in the antimicrobial function of GZA0 (Supplementary Figure S2D).

In order to further understand the PGP mechanism of strain GZY0, whole-genome sequencing was performed and genes related to PGP function were identified. The results showed that genes regulating the biosynthesis of IAA compounds (e.g., trpA, trpB, and trpC) were presented in the GZY0 genome and were involved in the synthesis of plant hormones. We hypothesized that GZY0 could directly promote plant growth and development by synthesizing IAA. GZY0 contained a variety of functional genes directly related to PGP (e.g., glnB, trkH, and trkA) that are involved in the synthesis of nitrogen and phosphorus, which are required for plant growth and development. We speculated that GZY0 may promote plant growth by improving the soil environment through nitrogen fixation and phosphorus solubilization. This strain contained some functional genes (e.g., yclQ and fetB) involved in the synthesis and transport of siderophores, which could increase the iron content in plants via iron chelation. The results indicated that these PGP-related genes in the genome of GZY0 may directly or indirectly promote plant growth (Supplementary Table S1).

The GZY0 genome also contained key genes promoting and inducing plant resistance. This included the functional genes lytE and ykfC, which regulate ABA biosynthesis. ABA can induce resistance to abiotic stress in plants and regulate physiological adaptation to abiotic stress. Additionally, we also identified key genes involved in the production of salicylic acid (pheT and pheS), which acts as a signaling molecule in the plant response to stress and can regulate important physiological and metabolic activities, strengthen the plant antioxidant defense system, and improve the stress resistance of plants (Supplementary Table S1).

The secondary metabolite synthesis gene clusters of GZY0 were predicted using antiSMASH. In total, 12 secondary metabolite synthesis gene clusters were predicted (Table 3), includes 3 NRPS, 1 RRE-containing, 1 PKS-like, 2 terpenes, 3 transAT-PKS, 1 T3PKS, and other types, and clear information was available for eight of them. Cluster1 (surfactin) contained 41 coding genes and shared 91% similarity with known gene clusters. Meanwhile, cluster2 (plantazolicin), cluster5 (macrolactin H), and cluster6 (bacillaene) contained 20, 44, and 44 coding genes and shared 91%, 100%, and 100% similarity with known gene clusters, respectively. Additionally, cluster7 (fengycin), cluster10 (difficidin), cluster11 (bacillibactin), and cluster12 (bacilysin) contained 66, 39, 45, and 42 coding genes, respectively, and shared 100% similarity with known gene clusters.

However, cluster3 (butirosin A/butirosin B), which contained 41 coding genes, only shared 7% similarity with known gene clusters. This suggested that this cluster in GZY0 may synthesize novel bacteriostatic substances and warrants further investigation. Meanwhile, three unknown secondary metabolite synthesis gene clusters (clusters 4, 8, 9) were detected in GZY0, of which two were linked to terpenes and one was linked to T3PKS. This showed that GZY0 may contain new antagonistic substance-related gene clusters. Additionally, these gene clusters comprise biosynthetic-additional, transport, regulatory, biosynthetic, and other genes (Supplementary Figure S3). This suggested that GZY0 could synthesize unique antibacterial substances to inhibit the growth of pathogens and indirectly promote plant growth through functional genes regulating the production of antimicrobial-related substances. Thus, GZY0 may have great potential applications in agricultural production.

Pangenome analysis of GZY0 and five reference strains using Orthofinder software yielded a total of 5,169 pan-genome entities, including 3,240 core genes accounting for 62.68%. The pan-genome size formula is y = 2,184.253 ^* x^**0.253 + 1,723.691. The parameter B (0.253) satisfies 0 < B < 1, indicating an open pan-genome. This suggests rich genetic diversity and the ability to rapidly adapt to environments by acquiring new genes (Figure 5A). The core gene formula is y = 1,656.261 ^* exp(−0.928 ^* x) + 3,250.146, where parameter B (−0.928) < 0. This indicates a closed-type core genome, suggesting all members share a stable genetic foundation (Figure 5B). Among the six Bacillus spp. strains, the number of core gene clusters was highest, followed by unique gene clusters, indicating genetic diversity and adaptive potential among strains (Figure 5C, Table 4). The six Bacillus spp. harbored 3,240 shared homologous genes. B. velezensis SQR9 possessed the highest number of unique homologous genes (301), while B. amyloliquefaciens DSM 7 had the fewest (4). GZY0 contained 171 unique homologous genes, suggesting it harbors numerous unique functions awaiting exploration (Figure 5D).

Compared to the control group (Figure 6A), A. konjac corms inoculated with P. aroidearum alone turned black and decayed significantly (Figure 6B). In comparison, A. konjac corm belonging to the GZY0 pretreatment group showed significantly lower morbidity (Figure 6C), and the rot control efficacy reached 45.81% (Table 5).