Bacillus velezensis BV01 Has Broad-Spectrum Biocontrol Potential and the Ability to Promote Plant Growth

To evaluate the potential of a bacterial strain as a fungal disease control agent and plant growth promoter, its inhibitory effects on phytopathogens such as Bipolaris sorokiniana, Botrytis cinerea, Colletotrichum capsici, Fusarium graminearum, F. oxysporum, Neocosmospora rubicola, Rhizoctonia solani, and Verticillium dahliae were investigated. The results showed that the inhibitory rates in dual-culture and sterile filtrate assays against these eight phytopathogens ranged from 57% to 83% and from 36% to 92%. The strain was identified as Bacillus velezensis based on morphological and physiological characterization as well as phylogenetic analyses of 16S rRNA and the gyrase subunit A protein (gyrA) regions. The results demonstrated that B. velezensis was able to produce fungal cell-wall-degrading enzymes, namely, protease, cellulase, and β-1,3-glucanase, and the growth-promotion substances indole-3-acetic acid (IAA) and siderophore. Furthermore, B. velezensis BV01 had significant control effects on wheat root rot and pepper Fusarium wilt in a greenhouse. Potted growth-promotion experiments displayed that BV01 significantly increased the height, stem diameter, and aboveground fresh and dry weights of wheat and pepper. The results imply that B. velezensis BV01, a broad-spectrum biocontrol bacterium, is worth further investigation regarding its practical applications in agriculture.


Introduction
Crop diseases caused by phytopathogens have resulted in a decrease in agricultural yields and quality, leading to significant economic losses [1].In particular, soil-borne fungal infections of important crops such as wheat, corn, rice, and pepper cause large economic losses [2].The United Nations 2030 Sustainable Development Goals suggested that the world should ensure sustainable consumption and production patterns, promote sustainable agriculture, and reduce environmental pollution [3].For a long time, synthetic chemical pesticides were commonly used in traditional agriculture to combat plant diseases, but they often caused environmental pollution and residual toxic effects in animals and humans [4].Thus, discovery of eco-friendly, long-lasting, and effective methods are required for disease prevention and management in agriculture.The use of microbial and biochemical agents has been explored as a practical alternative approach [5].
The plant-growth-promoting rhizobacteria (PGPRs) are often used for the production of bioactive substances that can protect plants by suppressing pathogens, inducing systemic resistance, or improving resistance to environmental stresses, by facilitating nutrient acquisition and modulating phytohormone levels in plants [6,7].In recent years, Bacillus subtilis and its closest relatives B. amyloliquefaciens, B. velezensis, B. cereus, and B. licheniformis have been widely used as biofertilizers and biofungicides [8,9].Bacillus velezensis FZB42, the classical PGPR strain, was successfully used as a biocontrol agent in potato, strawberry, wheat, and cabbage [10][11][12][13][14].The most prevalent plant fungal diseases, such as grey mold, Fusarium head blight, anthracnose, and root rot, etc., are mainly caused by species of Botrytis, Fusarium, Colletotrichum, and Rhizoctonia [15].This can be attributed to their broad host range, genetic diversity, rapid adaptation to plant disease resistance, and production of toxins [16].Previous studies have shown that B. velezensis is a promising agent for control of Rhizoctonia solani [17], Gaeumannomyces graminis var.tritici [18], Fusarium oxysporum f. sp.niveum [19], Botrytis cinerea, Colletotrichum gloeosporioides, and Phytophthora infestans [20], and it has attracted widespread attention in agricultural disease research.Nevertheless, studies on its biocontrol mechanism, screening of excellent strains, analyses of transcriptomics, proteomics, metabolomics, and research on industrial and commercial applications of B. velezensis are needed [21].
In this study, we aimed to assess the potential of a newly isolated bacterial strain, B. velezensis BV01, as a broad-spectrum biocontrol agent and investigate its capacity to control plant diseases and promote wheat and pepper development.The findings are of great significance for reducing the use of chemical fungicides to control soil-borne fungal diseases, thereby improving the ecological environment, and for providing technical support for food safety and sustainable development.

Tested Strains
Information on the 12 bacterial and fungal strains used is listed in Table 1.Bacillus velezensis BV01 was isolated from a contaminated potato dextrose agar (PDA) plate in the laboratory.Bacillus velezensis JDF and B. subtilis L01 and BS208 were isolated from three commercially available bacterial agents NongBaoShengWu ® , LvLong ® , and GuanLan ® , respectively, and the eight fungal plant pathogen strains were provided by colleagues from Beijing Academy Agriculture and Forestry Sciences, China Academy Agricultural Sciences, Guangxi Academy Agricultural Sciences, Nanjing Agricultural University, and our institute (Table 1).All strains were deposited in the China General Microbiological Culture Collection Center (CGMCC) and the State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences.

Evaluation of Antagonistic Abilities In Vitro
All bacterial strains were tested for their antagonistic abilities against eight phytopathogens in dual-culture assays [18].The bacterial strains were grown in nutrient broth (NB: peptone 10.0 g, beef paste 3.0 g, NaCl 5.0 g, dH 2 O 1000 mL) at 28 • C for 2 d, and the cell suspension was adjusted to 5 × 10 8 CFU/mL.The fungal strains were grown on PDA at 25 • C for 5 d.Mycelial plugs (5 mm in diam.) were placed in the center of each new PDA plate (90 mm in diam.);then, four sterile filter papers (5 mm in diam.), which contained 6 µL of a bacterial suspension, were placed evenly around individual mycelial plugs.Sterile water was used as the control.All plates were incubated at 25 • C for 5 d.Each treatment had three replicates.Measurements were calculated with the following formula: y = (A − B)/A × 100% (y: percentage of inhibition, A: colony diameter of pathogen on control plate, and B: colony diameter of pathogen in experimental group) [22].

Assessment of Antifungal Activity of Bacterial Sterile Filtrate
The antifungal activity of bacterial sterile filtrate was evaluated by measuring the diameter of the fungal colony [23].Four bacterial strains were incubated in NB with shaking at 180 rpm at 28 • C for 2 d, and then the cultures were centrifuged at 8000 rpm at 4 • C for 20 min to collect the supernatant.The supernatant was filtered through a 0.22 µm filter and then mixed into molten PDA at 45-50 • C with a concentration of 20% (v/v).The mycelium plug (5 mm in diam.) of each pathogen was placed at the center of each PDA plate with bacterial filtrate and incubated for 5 d at 25 • C. The PDA plate without bacterial filtrate was used as the control.Three replicates were set up for each treatment.The inhibition percentage (y%) was calculated with the following formula: y = (A − C)/A × 100% (A: growth radius of pathogen in control and C: growth radius of pathogen in different treatments).

Morphological Observation and Molecular Identification of Strain BV01
The morphological characteristics of strain BV01 were recorded after incubation on a nutrient agar (NA: peptone 10.0 g, beef paste 3.0 g, NaCl 5.0 g, agar 18.0 g, dH 2 O 1000 mL) plate at 28 • C for 2 d.A single colony was then taken and evenly spread onto a glass slide, dry fixed, and Gram stained for 1 min [24].The microscopic photographs were taken with a Zeiss AxioCam MRc 5 digital camera (Carl Zeiss, Jena, Germany) attached to Zeiss Axioskop 2 plus microscope (Carl Zeiss, Göttingen, Germany).
Genomic DNA was extracted from fresh cultures using a bacterial genomic DNA kit (ZOMAN, Beijing, China) according to the manufacturer's instructions.The 16S rRNA and the gyrase subunit A protein (gyrA) regions were amplified using the primer pairs of 27F (5 -AGAGTTTGATCCTGGCTCAG-3 ) and 1492R (5 -GGTTACCTTGTTACGACTT-3 ) and gyrA-F (5 -CAGTCAGGAAATGCGTACGTCTT-3 ) and gyrA-R (5 -CAAGGTAAT GCTCCAGGCATTGCT-3 ), respectively.PCR reactions were conducted using an ABI 2720 Thermal Cycler (Applied Biosciences, Foster City, CA, USA), and the products were then sequenced using an ABI3730XL DNA Sequencer (Applied Biosciences, Foster City, CA, USA).The newly obtained sequences and those of the ex-type strain as well as the related ones retrieved from GenBank were aligned using BioEdit 7.2 [25] and analyzed with the neighbor-joining (NJ) method [26] via MEGA X [27].The topological confidence of the resulting tree and the statistical supports of the branches were tested using the neighbor-joining bootstrap proportion (NJBP) with 1000 replications, each with 10 replicates of random addition of taxa [28].

Determination of Enzyme Activity and Secondary Metabolites
Protease, cellulase, and β-1,3-glucanase were detected on skim milk, carboxymethyl cellulose (CMC), and glucose agar, respectively [29].Siderophore and indole-3-acetic acid (IAA) were tested using modified chrome azurol S and Salkowski reagent agar, respectively [30].Enzyme activity and production of metabolites were observed based on the presence of clear zones around bacterial colonies after incubation at 28 • C for 3 d.All treatments were repeated three times.

Biocontrol Activity of Strain BV01 In Vivo
After evaluation of the antagonistic abilities of the four bacterial strains, two strains, namely, JDF and BV01, were further tested for their biocontrol and plant-growth-promotion abilities in a pot experiment.Four germinated wheat seeds were sowed per pot containing a mixture of podsolic soil and vermiculite (v:v = 1:1).On the 14th day, the roots of the plants were punctured and inoculated nearby the wounds with pathogen mycelium blocks (5 mm in diam.).After 24 h, the roots were irrigated with 30 mL (2.5 × 10 8 CFU/mL) of bacterial suspensions per wheat plant, and the same amount of NB was used as the control.The plants were kept in a greenhouse with a 14 h/10 h photoperiod/dark period at 26 ± 1 • C. The incidence of disease of wheat treated with BV01, JDF, and the non-treated control was recorded and calculated at 20 d after B. sorokiniana inoculation.Infection types (ITs) of B. sorokiniana on wheat were evaluated based on the area of a brown or black lesion at the stem base, with scores varying from 0 to 4 (IT0: no lesion, IT1: coverage of necrotic lesion less than 1/4, IT2: lesion coverage between 1/4 and 1/2, IT3: coverage between 1/2 and 2/3, and IT4: coverage between 2/3 and total).The disease index = ∑ (d i × l i ) / (L × di max ) × 100% (d i : infection type, li: number of plants with each infection type, and L: number of wheat plants investigated) [31].
Three true leaves at the age of 40 d were detached from pepper seedlings and punctured in two places with a sterile needle on both sides of the leaf.Mycelium blocks (5 mm in diam.) of F. graminearum were placed on the two wounds and then sprayed with 2 mL of either BV01 or JDF fermentation broth at a concentration of 2.5 × 10 8 CFU/mL, and the same volume of NB was used as the non-inoculated control at 24 h after F. graminearum inoculation.The treatments were kept in a greenhouse at 26 ± 1 • C with a 14 h/10 h photoperiod/dark period for 6 d; sterile water was added once to keep the treatment moist.There were 3 leaves in each pot and 3 replicates in each treatment.The diameters of diseased spots were recorded and calculated at the 6th day after F. graminearum inoculation.The inhibition rate was calculated as follows: (D CK − D i )/D CK × 100% (D CK : the control group's average colony diameter and Di: the treatment group's average colony diameter).

Plant-Growth-Promoting Assays in a Greenhouse
Sterilized wheat and pepper seeds were soaked in suspensions of BV01 or JDF at a concentration of 2.5 × 10 8 CFU/mL and then in sterile water for 10 min.NB was used as the control.The plants were kept in a greenhouse with a 14 h/10 h photoperiod/dark period at 26 ± 1 • C. The wheat and peppers were harvested at the 21st and 35th days, respectively.Plant height, fresh weight, dry weight, and leaf width were recorded, and the strong seedling index (SSI) was calculated [32].

Statistical Analysis
Statistical analysis was performed using SPSS 21 (Armonk, NY, USA).ANOVA was performed, and mean values were compared using Duncan's multiple range test with p < 0.05 as the level of significance.All analyses were conducted using GraphPad Prism 8 (San Diego, CA, USA).

Inhibitory Effects of Four Tested Bacterial Strains against Eight Fungal Phytopathogens
Strain BV01 exhibited varying degrees of antagonism against different phytopathogens, and the inhibition rates ranged from 57% to 83% (Table 2) with the highest potential inhibitory effects against B. cinerea PP1 (Figure 1, Treatment 1).The inhibition rates showed that BV01 had significantly higher inhibitory effects than JDF, L01, and BS208 on six of the eight tested fungal phytopathogens.The inhibition rates (%) (n = 3, mean ± SE).Different letters indicate significantly different groups (p < 0.05, ANOVA, Tukey HSD).

Inhibitory Effects of Four Tested Bacterial Strains against Eight Fungal Phytopathogens
Strain BV01 exhibited varying degrees of antagonism against different phytopathogens, and the inhibition rates ranged from 61% to 83% (Table 2) with the highest potential inhibitory effects against B. cinerea PP1 (Figure 1, Treatment 1).The inhibition rates showed that BV01 had significantly higher inhibitory effects than JDF, L01, and BS208 on six of the eight tested fungal phytopathogens.Antifungal assay by fermentation broth test showed that BV01 had relatively high inhibitory effects against different pathogens (Table 2), and the highest inhibitory rate reached 92% (against B. sorokiniana PP12) (Figure 1, Treatment 2).Overall, the effects of BV01 were better than those of JDF and L01 and were significantly superior to those of BS208.

Identification of Strain BV01
The colony of BV01 was ivory white and non-transparent with a rough surface on NA medium (Figure 2A-C).The cells were Gram-positive (Figure 2D), rod-shaped, 1.43-2.53µm long, 0.66-0.88µm wide, and occurred singly, in pairs, or occasionally in short Antifungal assay by fermentation broth test showed that BV01 had relatively high inhibitory effects against different pathogens (Table 2), and the highest inhibitory rate reached 92% (against B. sorokiniana PP12) (Figure 1, Treatment 2).Overall, the effects of BV01 were better than those of JDF and L01 and were significantly superior to those of BS208.

Identification of Strain BV01
The colony of BV01 was ivory white and non-transparent with a rough surface on NA medium (Figure 2A-C).The cells were Gram-positive (Figure 2D), rod-shaped, 1.43-2.53µm long, 0.66-0.88µm wide, and occurred singly, in pairs, or occasionally in short chains.The analysis of 16S rRNA sequences showed that strain BV01 shared 99% identity with the type strain of B. velezensis (CR502) according to a BLAST search.The resulting NJ trees based on sequences of 16S rRNA and gyrA (Figure 3) showed that BV01 clustered with Bacillus species and grouped with the type strain of B. velezensis, which confirmed its taxonomic position.
chains.The analysis of 16S rDNA sequences showed that strain BV01 shared 99% identity with the type strain of B. velezensis (CR502) according to a BLAST search.The resulting NJ trees based on sequences of 16S rRNA and gyrA (Figure 3) showed that BV01 clustered with Bacillus species and grouped with the type strain of B. velezensis, which confirmed its taxonomic position.chains.The analysis of 16S rDNA sequences showed that strain BV01 shared 99% identity with the type strain of B. velezensis (CR502) according to a BLAST search.The resulting NJ trees based on sequences of 16S rRNA and gyrA (Figure 3) showed that BV01 clustered with Bacillus species and grouped with the type strain of B. velezensis, which confirmed its taxonomic position.

Detection of Antagonism-Related Lytic Enzymes
Clear zones detected around the colony of BV01 indicated that the strain produced protease, cellulase, and β-1,3-glucanase (Figure 4A-C) as well as siderophore (Figure 4D) and IAA (Figure 4E), which suggested its high potential in biological control.The production of IAA reached 12.17 mg/mL after incubation for 6 d.

Detection of Antagonism-Related Lytic Enzymes
Clear zones detected around the colony of BV01 indicated that the strain produ protease, cellulase, and β-1,3-glucanase (Figure 4A-C) as well as siderophore (Figure and IAA (Figure 4E), which suggested its high potential in biological control.The prod tion of IAA reached 12.17 mg/mL after incubation for 6 d.

Biocontrol Effects of Bacterial Strains BV01 and JDF on Wheat Root Rot
Lesions at the stem bases of wheat were obviously brown in the non-treated con while those treated with BV01 and JDF were very slightly infected (Figure 5A).The dise indices of CK, BV01, and JDF were 76.4, 40.8, and 53.6, respectively (Figure 5B).In BV01 treatment, infection with wheat root rot was significantly (p < 0.05) reduced, the ative control efficacy was 47% (Figure 5C), and the fresh and dry weights (Figure 5D) plant height (Figure 5E) were increased by 24%, 91%, and 34%, respectively.

Biocontrol Effects of Bacterial Strains BV01 and JDF on Wheat Root Rot
Lesions at the stem bases of wheat were obviously brown in the non-treated control, while those treated with BV01 and JDF were very slightly infected (Figure 5A).The disease indices of CK, BV01, and JDF were 76.4, 40.8, and 53.6, respectively (Figure 5B).In the BV01 treatment, infection with wheat root rot was significantly (p < 0.05) reduced, the relative control efficacy was 47% (Figure 5C), and the fresh and dry weights (Figure 5D) and plant height (Figure 5E) were increased by 24%, 91%, and 34%, respectively.

Biocontrol Effect of Strain BV01 on Fusarium Wilt
The symptoms on pepper leaves of the control were severe, on those treated with JDF were moderate, and on those treated with BV01 were weak (Figure 6A).The average diameter of a spot was 2.31, 0.99, and 1.76 cm in the CK, BV01, and JDF treatments (Figure 6B).The control effect reached 57% and 24% for the treatments with BV01 and JDF, respectively (Figure 6C).

Biocontrol Effect of Strain BV01 on Fusarium Wilt
The symptoms on pepper leaves of the control were severe, on those treated with JDF were moderate, and on those treated with BV01 were weak (Figure 6A).The average diameter of a spot was 2.31, 0.99, and 1.76 cm in the CK, BV01, and JDF treatments (Figure 6B).The control effect reached 57% and 24% for the treatments with BV01 and JDF, respectively (Figure 6C).

Growth-Promotion Effects of Strain BV01 on Wheat and Pepper
Wheat treated with BV01 exhibited an increase in height of 13% (Figure 7A), while the fresh weight and dry weight were improved by 10% and 5% (Figure 7B).Pepper treated with BV01 exhibited increases in the fresh weight, leaf width, and stem thickness of 20%,

Growth-Promotion Effects of Strain BV01 on Wheat and Pepper
Wheat treated with BV01 exhibited an increase in height of 13% (Figure 7A), while the fresh weight and dry weight were improved by 10% and 5% (Figure 7B).Pepper treated with BV01 exhibited increases in the fresh weight, leaf width, and stem thickness of 20%, 9%, and 9%, respectively.The dry weight and plant height were improved by 12% and 2% (Figure 7C,D).The SSI for treatment with BV01 and JDF increased by 19% and 10%.These findings suggest that strain BV01 was more effective in promoting plant growth than JDF.

Discussion
For a long time, Bacillus amyloliquefaciens and B. subtilis were known to have biocontrol functions against various plant pathogens [33].Recently, B. velezensis was reported as a biocontrol agent against many phytopathogens.For example, B. velezensis strain F21 can control Fusarium wilt on watermelon [19], and strain BR-01 has strong antagonistic effects on rice pathogens [34], while strain CE100 increases fruit yield of strawberries by controlling fungal diseases [35].The star strain FZB42 was initially established in 1998, and successive studies on its antimicrobial substances, interactions between plants and bacteria, regulatory small RNAs, and biocontrol enzymes have been carried out [33].In previous studies, antagonistic strains of B. velezensis were often isolated from water, soil, air, plant roots, plant surfaces, and animal intestines [7].In the present study, strain BV01 was derived from a PDA plate in the laboratory and speculated to be an air source strain.Based on morphological characteristics and phylogenetic evidence, strain BV01 was identified as B. velezensis; further exploration of its biological control potential was then performed.Its dual-culture inhibition rates against different pathogens were greater than 56%, and the fermentation broth inhibition rates were reduced by more than 36% when compared to the control.The results indicate that BV01 produces a special antibacterial substance.Some lipopeptide extract components of B. amyloliquefaciens have been demonstrated as key substances in controlling the growth of Xanthomonas citri subsp.citri [36].Zhou et al. [34] proved that the relative inhibition rate of B. velezensis BR-01 against F. fujikuroi was

Discussion
For a long time, Bacillus amyloliquefaciens and B. subtilis were known to have biocontrol functions against various plant pathogens [33].Recently, B. velezensis was reported as a biocontrol agent against many phytopathogens.For example, B. velezensis strain F21 can control Fusarium wilt on watermelon [19], and strain BR-01 has strong antagonistic effects on rice pathogens [34], while strain CE100 increases fruit yield of strawberries by controlling fungal diseases [35].The star strain FZB42 was initially established in 1998, and successive studies on its antimicrobial substances, interactions between plants and bacteria, regulatory small RNAs, and biocontrol enzymes have been carried out [33].In previous studies, antagonistic strains of B. velezensis were often isolated from water, soil, air, plant roots, plant surfaces, and animal intestines [7].In the present study, strain BV01 was derived from a PDA plate in the laboratory and speculated to be an air source strain.Based on morphological characteristics and phylogenetic evidence, strain BV01 was identified as B. velezensis; further exploration of its biological control potential was then performed.Its dual-culture inhibition rates against different pathogens were greater than 56%, and the fermentation broth inhibition rates were reduced by more than 36% when compared to the control.The results indicate that BV01 produces a special antibacterial substance.Some lipopeptide extract components of B. amyloliquefaciens have been demonstrated as key substances in controlling the growth of Xanthomonas citri subsp.citri [36].Zhou et al. [34] proved that the relative inhibition rate of B. velezensis BR-01 against F. fujikuroi was 57%, while the strain showed no antagonistic ability against R. solani.The results of the current study revealed that strain BV01 possessed very strong antagonistic activity and broadspectrum biological ability against B. cinerea, F. oxysporum, C. capsici, V. dahliae, R. solani, B. sorokiniana, F. graminearum, and N. rubicola.
Many Bacillus species produce a variety of hydrolytic enzymes, such as cellulase, β-1,3glucanase, and protease, which are responsible for the degradation of diverse components of fungal pathogens [35,37].The detection of cellulase, protease, and β-1,3-glucanase in BV01 supports its association with the growth suppression of several fungal phytopathogens.Our results also revealed that strain BV01 effective in vitro against fungal pathogens was also able to produce siderophores, which are related to indirect antagonistic processes such as plant defenses and growth promotion [30].Moreover, some members of Bacillus invade the rhizosphere of plants and promote plant growth by producing plant hormones, such as IAA, cytokinins, and gibberellins, and chelating minerals and siderophores.Many plant-growth-promoting bacteria produce IAA, which promotes the development of plant roots, and are usually utilized as bioinoculants [38][39][40][41][42][43][44][45].In a previous study, B. velezensis BY6 was reported to significantly increase the dry and fresh mass and plant height of Pdpap poplar seedlings [46].In the present study, B. velezensis BV01 produced IAA during its growth.Moreover, our pot experiment results revealed that pepper and wheat treated with strain BV01 possessed higher fresh weight, dry weight, plant height, leaf width, stem thickness, and SSI than controls.Both the antifungal activity assay and greenhouse pot experiment indicated that the strain BV01 has biocontrol and plant-growth-promotion potential.
Wheat and pepper are two of the most commonly grown crops and vegetables in the world.Several pathogens cause severe diseases of them and thus reduce significantly their yields.For example, wheat root rot caused by B. sorokiniana, Fusarium spp., and other pathogens alone or in combination generally can lead to wheat yield reductions of 20%-30%, with severe cases of more than 50% [47,48].Previous studies revealed that B. subtilis and B. amyloliquefaciens can prevent and control wheat root rot [47].However, there are few studies on the effects of B. velezensis on wheat root rot caused by B. sorokiniana.Bacillus velezensis strains CC09 and NEAU-242-2 could be used as potential biocontrol agents to control wheat disease [49,50].In this study, B. velezensis strain BV01 was able to effectively control wheat root rot caused by B. sorokiniana in a greenhouse, with a control rate of 47%.The occurrence of pepper wilt is increasing currently and seriously affects the quality of pepper.For example, the incidence of pepper wilt disease in China is generally 15%-30%, with severe cases decreasing quality by 70%-80% [51].The main pathogen, Fusarium graminearum, is a highly destructive phytopathogen, not only lowering crop yields but also producing mycotoxins and affecting crop quality.Previous studies have confirmed that B. velezensis could control pepper root rot [52], wheat spikes [53], corn stalk rot [54], and corn head blight [55].To our knowledge, the present study is the first report that B. velezensis can serve as a potential biocontrol agent for controlling pepper wilt induced by F. graminearum.Bacillus velezensis BV01 not only promotes the growth of wheat and pepper seedlings but also significantly controls wheat root rot and pepper wilt.In summary, Bacillus velezensis BV01 has good control effects in both dual-culture and fermentation broth tests against B. sorokiniana and F. graminearum, and it obviously reduced the disease symptoms and promoted the growth of wheat and pepper.

Conclusions
Bacillus velezensis BV01 showed protease, cellulase, and β-1,3-glucanase activities, which are related to phytopathogen cell wall degradation, and produced growth-promotion substances such as IAA and siderophore.This strain also suppressed the growth of eight phytopathogens both in dual-culture and sterile filtrate assays and significantly reduced the disease incidence of wheat root rot and Fusarium wilt in greenhouse settings.Moreover, it significantly promoted wheat and pepper growth.In conclusion, BV01 exhibits broad and effective antagonistic activity against several phytopathogens, promotes plant growth, and is worthy of further exploration of its biocontrol applications in eco-friendly agriculture practices.

Figure 1 .
Figure 1.Inhibitory effects of BV01 against fungal phytopathogens.CK: only pathogen on PDA at 25 °C for 5 d; Treatment 1: dual culture of BV01 against pathogen on PDA at 25 °C for 5 d; Treatment 2: pathogen on PDA amended with fermentation broth of BV01 at 25 °C for 5 d.

Figure 1 .
Figure 1.Inhibitory effects of BV01 against fungal phytopathogens.CK: only pathogen on PDA at 25 • C for 5 d; Treatment 1: dual culture of BV01 against pathogen on PDA at 25 • C for 5 d; Treatment 2: pathogen on PDA amended with fermentation broth of BV01 at 25 • C for 5 d.

Figure 3 .
Figure 3. Phylogenetic trees generated based on sequences of 16S rRNA (A) and gyrA (B) regions of Bacillus species.NJBP values greater than 75% are shown at the nodes.

Figure 3 .
Figure 3. Phylogenetic trees generated based on sequences of 16S rRNA (A) and gyrA (B) regions of Bacillus species.NJBP values greater than 75% are shown at the nodes.

Figure 3 .
Figure 3. Phylogenetic trees generated based on sequences of 16S rRNA (A) and gyrA (B) regions of Bacillus species.NJBP values greater than 75% are shown at the nodes.

Figure 5 .
Figure 5. Inhibitory effect of Bacillus strains on wheat root rot disease caused by B. sorokiniana PP12.(A) Symptoms of B. sorokiniana on wheat roots with different treatments; (B) disease index of B. sorokiniana in the treatments with BV01 and JDF; (C) inhibition rates of BV01 and JDF; (D) fresh and dry weights of wheat; (E) shoot biomass (cm) measured by plant height of wheat.The results were observed after 40 d of incubation.Values are the means ± SEs, n = 27 plants, ** p < 0.001, and * p < 0.05.

Figure 5 . 14 Figure 6 .
Figure 5. Inhibitory effect of Bacillus strains on wheat root rot disease caused by B. sorokiniana PP12.(A) Symptoms of B. sorokiniana on wheat roots with different treatments; (B) disease index of B. sorokiniana in the treatments with BV01 and JDF; (C) inhibition rates of BV01 and JDF; (D) fresh and dry weights of wheat; (E) shoot biomass (cm) measured by plant height of wheat.The results were observed after 40 d of incubation.Values are the means ± SEs, n = 27 plants, ** p < 0.001, and * p < 0.05.Microorganisms 2023, 11, x FOR PEER REVIEW 9 of 14

Figure 6 .
Figure 6.Effect of Bacillus strains on disease symptoms caused by F. graminearum PP15 on leaves.(A) Symptoms of F. graminearum on leaves with different treatments; (B) spot diameter after treatment with BV01 or JDF; (C) inhibition rates of BV01 and JDF.Values are the means ± SEs, n = 9 leaves, ** p < 0.001, and * p < 0.05.

Figure 7 .
Figure 7. Growth-promotion effects of BV01 and JDF on wheat and pepper.Wheat growth (A) and experimental conditions (B) in CK, BV01, and JDF pot experiments, n = 12 plants.Pepper growth (C) and experimental conditions (D) in CK, BV01, and JDF pot experiments, n = 9 plants.Values are the means ± SEs, different letters show significant differences (Fisher's LSD, p < 0.05).

Table 1 .
The bacterial and fungal strains tested in this study.