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Article

A Promising Biocontrol Agent of Bacillus velezensis VC3 against Magnaporthe oryzae and Colletotrichum gloeosporioides in Plants

by
Kui Li
1,2,*,†,
Xin Zhang
3,4,5,†,
Yue Chen
3,4,
Tong Zhang
3,
Qianlong Sun
3,
Hongtao Wu
6,
Zhen Chen
1,2,
Yan Sun
6,
Dehua Liu
1,2 and
Xinqiu Tan
3,4,5,*
1
Key Laboratory of Industrial Biocatalysis (Ministry of Education), Department of Chemical Engineering, Tsinghua University, Beijing 100084, China
2
Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, China
3
Hunan Plant Protection Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China
4
Longping Branch, College of Biology, Hunan University, Changsha 410125, China
5
Yuelushan Laboratory, Changsha 410082, China
6
Jiangsu Tsingda Smart Biotech Co., Ltd., Taicang 215400, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Processes 2024, 12(7), 1490; https://doi.org/10.3390/pr12071490
Submission received: 16 May 2024 / Revised: 19 June 2024 / Accepted: 19 June 2024 / Published: 16 July 2024
(This article belongs to the Section Biological Processes and Systems)
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Abstract

:
Fungal diseases of plants are one of the key factors causing global crop losses. In this study, we isolated a Bacillus velezensis strain VC3, which was found to have a broad-spectrum inhibitory effect on a variety of phytopathogenic fungi through in vitro and in planta experiments, especially on Magnaporthe oryzae and Colletotrichum gloeosporioides. Further genomic and transcriptomic analyses revealed that the B. velezensis VC3 has multiple functional gene clusters encoding for the synthesis of a variety of antifungal secondary metabolites, including antimicrobial peptides (AMPs) and lipopeptides (LPs). In addition, AMPs and LPs were isolated and purified from B. velezensis VC3 fermentation broth and their antifungal activities were verified in this study. AMPs and LPs significantly inhibited spore germination, appressorium formation, and disease development, and AMPs have a better potential for controlling M. oryzae and C. gloeosporioides than LPs. These findings open new avenues for utilizing B. velezensis VC3 as biocontrol agents, providing potential sustainable solutions for agricultural production.

1. Introduction

Plant diseases are a significant factor contributing to a decrease in crop yield, with fungal diseases particularly impactful. Data from the Food and Agriculture Organization of the United Nations (FAO) indicates that plant diseases result in a 14% loss in global crop production annually, with fungal diseases alone responsible for 42% of this loss [1]. The current reliance of the agricultural industry on chemical fungicides to combat fungal diseases raises concerns due to the adverse effects associated with these chemical agents, including chemical residues, environmental pollution, and the development of pest resistance [2]. In light of these challenges, there is a growing recognition of the need for sustainable agricultural practices, prompting the exploration of alternative strategies such as the use of microorganisms for biocontrol and biofertilization to uphold crop productivity and safety while reducing dependence on chemical inputs [3].
The adoption of biological control agents, such as Bacillus isolates, is gaining momentum as a sustainable approach to managing plant diseases in agricultural systems [4,5]. Notably, recent studies have highlighted the potential of specific Bacillus strains, including B. subtilis isolate G5 inhibiting M. oryzae conidia formation [6] and B. velezensis effectively suppressing C. gloeosporioides and protecting orange fruits during post-harvest stages [7]. Given the complexities of field conditions, there is a need to deepen our understanding and resources pertaining to biocontrol strains targeting M. oryzae and C. gloeosporioides to expand the range of effective biocontrol agents and elucidate their mechanisms of action.
Among the various bacterial antagonists reported, Bacillus spp. like Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus velezensis have been widely used as biofungicides by producing a variety of biologically active compounds with a broad spectrum of activities toward phytopathogens [8,9]. Previous studies have shown that B. velezensis is a promising agent for the control of Phytophthora infestans [10], Rhizoctonia solani [11], Colletotrichum orbiculare, and Fusarium oxysporum [12]. B. velezensis has the ability to protect plants against pathogens and can promote plant growth [13]. Nevertheless, further studies on its biocontrol mechanism, analyses of transcriptomics, proteomics, and research on industrial and commercial applications of B. velezensis are needed [14].
Rice blast and pepper anthracnose are important fungal diseases causing significant annual losses to global rice or pepper production [15]. They are caused by the filamentous fungus Magnaporthe oryzae and Colletotrichum gloeosporioides, separately. Conidia of M. oryzae and C. gloeosporioides play a central role in the disease cycle. Conidia can spread through wind, rainwater, or human activities, and when attached on the host surface, conidia begin to germinate and develop dome-shaped infection cells called appressoria from the end of the germ tubes [16,17]. Currently, the control of these two diseases is mainly based on breeding and utilizing disease-resistant varieties along with supplementation by chemical fungicides. Therefore, exploring a practical alternative approach, such as the use of microbial and biochemical agents, to control these two diseases is of great significance for ensuring food security [18]. Although recent studies have investigated the biological control of M. oryzae and C. gloeosporioides by Bacillus isolates, revealing that B. subtilis isolate G5 inhibits the formation of conidia of M. oryzae [6] while culture suspensions of B. velezensis could strongly inhibit C. gloeosporioides and shield orange fruits at post-harvest stages [7], the biological control mechanism for these two diseases is still understudied.
In this study, a biocontrol B. velezensis strain VC3 with broad-spectrum inhibition of plant pathogens was isolated, and by conducting in vitro and greenhouse experiments to inhibit the pathogens, it was determined that strain VC3 has the potential to be used as a biocontrol agent to control M. oryzae and C. gloeosporioides in agriculture. The discovery of B. velezensis VC3’s broad-spectrum efficacy against a multitude of plant pathogens offers a versatile solution to combat various fungal infections that threaten agricultural productivity. In addition, our research delves into the antifungal mechanisms employed by VC3 against the devastating pathogens M. oryzae and C. gloeosporioides, revealing strategies that specifically target the critical stages of fungal infection, such as spore germination and appressorium formation. The possible biocontrol mechanisms of strain VC3 were then explored by mining genomic and transcriptomic information. We then isolated and purified antimicrobial peptides (AMPs) and lipopeptides (LPs) from the VC3 fermentation broth and resolved the antagonistic mechanisms of the two classes of compounds against M. oryzae and C. gloeosporioides. This study is expected to provide a solid foundation for the development and application of biocontrol agents of B. velezensis.

2. Material and Methods

2.1. Microorganisms, Plants, and Growth Conditions

The B. velezensis strain VC3 was isolated from the rhizosphere of a hyperaccumulating nickel plant (Mosiera bullata) in an ultramafic reservoir of the Cuenca de Cubanacán, Villa Clara, Cuba. The strain VC3 was deposited in the China General Microbiological Culture Collection Center with the deposition number CGMCC No. 28737. The VC3 was cultured in Lysogeny Broth (LB; 5 g L−1 tryptone, 10 g L−1 yeast extract, and 10 g L−1 NaCl) medium with 20 g L−1 glucose, and the liquid cultures were shaken for 24 h at 37 °C at 200 rpm.
Eight fungal strains used to test the antimicrobial activity of B. velezensis VC3 were isolated and preserved by Hunan Plant Protection Institute, Hunan Academy of Agricultural Sciences. These fungal strains were Magnaporthe oryzae, Colletotrichum gloeosporioides, Sclerotinia sclerotiorum, Phytophthora capsici, Exserohilum mrcicum, Fusarium oxysporum, Rhizoctonia solani, and Selerotium rolfsii. These fungi were grown on Potato Dextrose Agar (PDA; 20 g L−1 glucose, 4 g L−1 potato extract, and 15 g L−1 agar) medium at 28 °C.
The rice variety was Co39, provided by Dr. Yue Chen, Hunan Plant Protection Institute, Changsha, China. The pepper variety was Xiangyan 15, purchased from Hunan Xiangyan seed industry Co., Ltd., Changsha, China.

2.2. In Vitro Effect on Mycelial Growth

To evaluate the antagonistic effect of B. velezensis VC3 on the mycelial growth of pathogenic fungi, a petri plate culture assay was performed. B. velezensis VC3 single colonies were taken with an inoculation needle and cultured in 5 mL LB with glucose medium at 37 °C for 24 h at 200 rpm. The fungal strains were cultured in PDA medium for four to seven days. A 6 mm mycelial plug was removed from the edge of an actively growing culture of each pathogenic fungus and placed upside down in the center of an aquantitative PDA medium plate. Four sterilized 1 mm thick sterile paper disks (6 mm in diameter) were placed on the medium equidistantly and 20 mm from the border of the plates. A drop of 20 μL of VC3 suspensions (prepared as described above) were pipetted onto each sterile paper disk. The plates of pathogenic fungi with LB + glucose medium instead of bacterial cell suspension were used as negative controls.
The plates were incubated in a 28 °C incubator and cultured for four to seven days. The inhibition rate (I) of mycelial growth was calculated as I(%) = [(D1 − D2)/D1] × 100. D1 is the diameter of the fungal colony of negative control, and D2 is the mycelium growth between two paper disks in the VC3 treatment. The experiments were performed in at least three biological replicates and repeated three times.

2.3. Sequencing and Annotation of the Strain VC3 Genome

VC3 strains cultured to the mid-logarithmic stage were centrifuged, and the cells were collected. The VC3 genome was extracted using a genomic DNA extraction kit (DP304-02, TIANGEN, Beijing, China). The extracted VC3 genome was broken using the ultrasonic method to obtain a fragment of about 500 bp. The sticky ends formed by the breakage were repaired to flat ends; then, the DNA fragment could be connected to the junction with a “T” base at the 3′ end by adding base “A”; and then, electrophoresis was used to recover the ligation product of the target fragment. The target fragment was recovered by electrophoresis; then, PCR was used to amplify the DNA fragments with junctions at both ends; and finally, the qualified library was used for cluster preparation and sequencing.
The genome sketch of VC3 was sequenced using the Illumina HiSeq platform sequencing technology (Azenta life science, Suzhou, China). The Kyoto Encyclopedia of Genes and Genomes database (KEGG, https://www.genome.jp/kegg/, accessed on 11 May 2022), Clusters of Orthologous Groups (COG, ftp://ftp.ncbi.nlm.nih.gov/pub/COG, accessed on 11 May 2022), Carbohydrate-Active enZYmes Database (CAZy, http://www.cazy.org/, accessed on 11 May 2022), Non-Redundant Protein Database (NR, https://www.ncbi.nlm.nih.gov/refseq/about/nonredundantproteins/, accessed on 11 May 2022), and gene ontology (GO, https://www.geneontology.org/, accessed on 11 May 2022) were used for predicting functional proteins. The assembly data of the VC3 genome were uploaded to the National Center for Biotechnology Information (NCBI) with the accession number PRJNA1043409.

2.4. Taxonomic Classification of the Strain VC3

A phylogenetic tree was constructed based on 16 S rRNA and 8 housekeeping gene sequences given in the Online Resource (Table S1). The phylogenetic tree was generated using the maximum-likelihood method by Molecular Evolutionary Genetics Analysis version 11 [19].

2.5. Determination of the Gene Expression Level of the Strain VC3

The strain VC3 was incubated in LB medium, and the samples were collected at 24 h and 48 h. Cells were collected, and total RNA was extracted using TRIzol reagent. The quality and quantity of RNA were assessed using a NanoDrop spectrophotometer and agarose gel electrophoresis. cDNA libraries were sequenced on an Illumina HiSeq 2000 platform sequencing technology (Azenta life science, Suzhou, China). The obtained reads were filtered and mapped to the reference genome using the HISAT2 algorithm. The mapped reads were assembled into transcripts using StringTie software (v.2.2.0; Baltimore, MD, USA) and compared with known genes using the BLAST algorithm to identify novel transcripts and splice variants.
Gene expression was calculated using the Fragments Per Kilo bases per Million reads (FPKM) method of the Htseq software (v.0.6.1; Sydney, Australia) [20].

2.6. Isolation of LPs and AMPs

The VC3 strain was incubated for 48 h at 37 °C with shaking at 200 rpm. The fermentation broth was centrifuged at 12,000× g for 20 min. The pH of the supernatant was adjusted to 2.0 with 6 M HCl and was then stored overnight at 4 °C. The sample was centrifuged again at 12,000× g for 20 min to harvest the solid crude LPs, and then, the solid crude LPs were washed 3 times with dd H2O (pH 1.8). The precipitate was resuspended with acetone. Insoluble impurities were removed by centrifugation. The acetone solution containing LPs were then placed in a fume hood for air-drying to obtain the dried LPs. The dry powder was dissolved with water, and the solution was filtered using a 0.22 μm filter to obtain a sterile lipopeptide solution for subsequent use. The LPs were characterized by Liquid Chromatography Mass Spectrometry (LC-MS), as previously reported [21].
The antimicrobial peptides (AMPs) were isolated and purified following the ammonium sulfate precipitation method. Briefly, ammonium sulphate was added to the VC3 culture supernatant until 20% saturation. The solution was kept overnight at 4 °C, and the precipitate was collected by centrifugation at 15,000× g at 4 °C for 20 min. A volume of 0.02 mol L−1 Tris buffer (pH 6.8) was used to dissolve the precipitate. Larger molecules of protein were removed by filtering the solution using 100, 30, and 10 KDa ultrafiltration tubes. AMPs were intercepted and salts were removed by filtration using 3 KDa ultrafiltration tubes. A sterile filtrate of AMP was obtained through a 0.22 um filter membrane. The AMP was then characterized by SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) and LC-MS/MS.

2.7. Effect on Conidia Gemination and Appressorium Formation of M. oryzae and C. gloeosporioides

The effect of cell-free filtrates and active constituents on conidia gemination and the appressorium formation of pathogenic fungi was assessed. B. velezensis VC3 suspensions (prepared as described above) were centrifuged at 8000 rpm for 10 min, and the supernatant was recovered and filtered through a syringe containing Millipore Millex-GV filters (0.22 µm pore size). The filtrates were stored at 4 °C until use. The active constituents were prepared as described above.
For the conidiation of M. oryzae, strain blocks were maintained on Straw Decoction and Corn (SDC) agar media at 28 °C for 7 days in the dark followed by 2 days of continuous illumination under fluorescent light [22]. Conidia were harvested in sterile distilled water with a sterile glass rod rubbing the hyphae. The conidia collected from C. gloeosporioides were isolated from the cultures on Complete Medium (CM) liquid media at 28 °C for 3 days [23]. Conidia suspensions were filtered through two layers of Miracloth (CALBIOCHEM) and resuspended to a concentration of 1 × 104 conidia per mL in sterile distilled water. Droplets (20 μL) of conidial suspension treated with cell-free filtrates (10%) or active constituents (final concentration of 30 μg/mL or 6 μg/mL) were placed on plastic cover slips and then incubated under humid conditions at 28 °C. The suspensions, which were treated with a medium of Bacillus or sterile distilled water, were used as a control. The percentages of conidia gemination were determined by microscopic observation after 2 and 4 h, and germinated conidia forming appressoria was observed at 6 h post-inoculation. Pictures of each were taken. The appressorium formation rates were calculated by dividing the number of conidia with appressoria by the number of germinated conidia. Three biological replicates were maintained, with each replicate containing at least 50 conidia. All experiments were repeated three times.

2.8. Plant-Growth-Promoting Assays in a Greenhouse

B. velezensis VC3 suspensions were prepared as described above. Sterilized rice and pepper seeds were soaked in suspensions of B. velezensis VC3 at a concentration of 1% or 0.5% for 24 h. The medium was used as the control. The germination of rice seeds was observed and recorded after 2 days of incubation. The plants were kept in a greenhouse with a 12 h/12 h photoperiod/dark period at 25 °C. The germination of the pepper seeds was observed and recorded on the 10th day. Ten milliliters of the diluted VC3 suspensions (1% or 0.5%) was applied per pot at 2-day intervals on 3- or 7-day-old rice or pepper seedlings. The rice and peppers were harvested on the 10th and 20th days, respectively. Plant height and root length were recorded. This experiment was performed three times, with at least 30 seedlings for each treatment.

2.9. In Planta Biological Control

The biological control of M. oryzae and C. gloeosporioides was tested on the leaves of the planted pepper, rice, and barley. Conidia suspensions of M. oryzae and C. gloeosporioides (prepared as described above) were adjusted to 1 × 105 conidia/mL.
For the spray inoculation test, 5 mL of an M. oryzae conidial suspension containing B. velezensis VC3 suspensions (10%) and gelatin (0.2%) was sprayed onto 2-week-old susceptible rice plants with a sprayer. For the detached leaf assay, the leaves from 8-day-old barley seedlings were used. A total of 10 μL of the dilution-drop conidia suspensions (1 × 105, 1 × 104, and 1 × 103 conidia/mL) containing gelatin (0.2%) and active constituents (final concentration of 30 μg/mL) was placed onto the upper side of the barley leaves maintained on 4% (w/v) water agar (WA) plates [24]. The inoculated plants were kept in a growth chamber at 25 °C with 90% humidity and in the dark for the first 24 h, followed by a 12/12 h light/dark cycle. The conidial suspensions treated with 0.2% gelatin were used as a control.
C. gloeosporioides infection assays were performed on detached pepper leaves. The seeds of pepper were transferred to pots until they grew to the fifth-to-sixth-leaf stages. Detached leaves with wound sites were inoculated with 20 μL of conidia suspension containing B. velezensis VC3 suspensions (10%) or active constituents (final concentration of 30 μg/mL) on the reverse side. Inoculated plants were kept at 28 °C with 90% humidity and in the dark for 2–4 days. The conidial suspensions were used as a control.
Lesion formation was observed daily and photographed 3–7 days after inoculation. The mean number of lesions formed on rice and the sizes of lesions formed on pepper and barley were measured using an image analysis system (Image-Pro Plus 6.0, Media Cybernetics, Inc., Rockville, MD, USA). This experiment was performed three times, with at least five replicates for each treatment.

2.10. Data Analysis

The statistical significance of the results was analyzed by two-way analysis of variance (ANOVA). The multiple Duncan test was used for comparing the differences between mean values at a significance level of p < 0.05. All analyses were run in the SPSS 20.0 software (SPSS Inc., Chicago, IL, USA).

3. Results

3.1. Inhibitory Effects of B. velezensis VC3 against Eight Fungal Phytopathogens

B. velezensis VC3 demonstrated varying degrees of antagonism against different fungal phytopathogens, exhibiting higher potential inhibitory effects against S. sclerotiorum, M. oryzae, and C. gloeosporioides (Figure 1). The inhibition rates were 81.34% against S. sclerotiorum, 59.28% against M. oryzae, and 53.39% against C. gloeosporioides (Table 1).

3.2. Effect of VC3 on Rice and Pepper Seedling Growth

Rice treated with VC3 exhibited unaffected growth in seed germination rate, height of seedling, and root length (Table S1), as compared with the medium control. The root length of peppers treated with VC3 improved between 17.6% and 41.2% compared with the control seedlings, while the seed germination rate and height of seedlings displayed the same levels (Table S1). These findings suggest that VC3 does not affect the seedling growth of rice or pepper.

3.3. Genome Analysis of Bacillus Velezensis VC3

In order to investigate the mechanism of fungal disease control by the VC3 strain, we sequenced the VC3 genome and obtained a sketch of the VC3 genome. The VC3 genome contains 3,882,010 bp bases with a GC content of 46.5%. The VC3 genome was predicted to have 3715 protein-coding genes, 83 tRNA genes, and 10 rRNA genes. When comparing the genomes of VC3, FZB42, and QST713, it was observed that the genome size of strain VC3 was similar to FZB42 (3,918,589 bp) but smaller than that of QST713 (4,233,757 bp) (Table 1). Additionally, the average of the G  +  C content of VC3 was similar to that of FZB42 (46.5%) and higher than that of QST713 (45.9%) (Table 1). Moreover, the genome of VC3 contained 91 pseudo genes, which was higher than the number found in FZB42 (59) and QST713 (68).
To determine the genetic relationships of VC3 with other strains in Bacillus, a phylogenetic tree was established based on the 16S rRNA gene, and eight housekeeping gene sequences—atpD, gyrA, gyrB, recN, topA, uvrC, ftsZ, and infB—among Bacillus strains. Based on the observed genetic distance relationships, strain VC3 was closely clustered together with B. velezensis FIO1408 (Figure 2A). The distribution of homologous species in the NR database showed that the highest percentage of homologous proteins originated from the genus Bacillus (Figure 2B).
Of the 3715 protein-coding genes in the VC3 genome, 2637 (71.0%) and 2921 (78.6%) were annotated using the KEGG and COG database, respectively (Figure 2C,E). Among these annotated genes in the COG database, 121 genes were classified and annotated as being involved in secondary metabolite biosynthesis, transport, and catabolism (Figure 2E). The genome of B. velezensis VC3 contained 432 CAZy genes, including 8 auxiliary activity (AA) genes, 69 carbohydrate-binding module (CBM) genes, 34 carbohydrate esterase (CE) genes, 108 glycoside hydrolase (GH) genes, 210 glycosyl transferase (GT) genes, and 3 polysaccharide lyase (PL) genes (Figure 2D).

3.4. Gene Clusters Involved in the Synthesis of Secondary Metabolites

Using the AntiSMASH database, nine putative secondary metabolite biosynthetic gene clusters (BGCs) were identified in the genome of VC3, covering 11.6% of the entire genome. Of these BGCs, three corresponded to non-ribosomal peptide synthetases (NRPS), four to polyketide synthases (PKS), one to dipeptide, and one to AMP (Table 2). Most of these compounds were associated with the production of NRPS and PKS systems. Among the nine BGCs, seven clusters were identified to play key roles in the synthesis of fengycin, surfactin, iturin, bacillibactin, bacillaene, difficidin, macrolactin H, and bacilysin (Table 1). The gene clusters responsible for synthesizing fengycin (fenABCDE), surfactin (srfAABCD), iturin (ituDABC), bacillibactin (dhbACEBF), bacillaene (baeBCDE,acpK, baeGHIJLMNR), difficidin (difABCDEFGHIJKLMNO), macrolactin H (mlnABCDEFGHI), and bacilysin (bacABCDEFG) in the genome of VC3 are 47,585 bp, 43,672 bp, 69,391 bp, 51,791 bp, 72,467 bp, 70,775 bp, 54,820 bp, and 41,419 bp long, respectively. Additionally, we identified one gene encoding AMPs in the genome of the VC3 strain.

3.5. Analysis of the Secondary Metabolite Gene Expression Levels in Bacillus Velezensis VC3

As shown in Table S1, gene expression was detected in 3539 (90.12%) and 3353 (85.38%) genes of VC3 after 24 h and 48 h of incubation, respectively, of which 1078 (30.46%) and 913 (27.23%) were highly expressed genes (FPKM > 60). An analysis of the relative expression levels of the antimicrobial gene clusters showed that the synthesized genes for AMP, surfactin, iturin, difficidin, bacillaene, and macrolactin H were highly expressed genes, whereas the fengycin, bacillibactin, and bacilysin gene clusters were relatively lowly expressed genes (Figure 3).

3.6. Isolation and Characterization of LPs and AMPs in B. velezensis VC3

The LPs were purified from the VC3 fermentation broth by acid precipitation, with lipopeptide concentrations of ~400 mg/mL at 72 h (Figure 4A). Three classes of LPs—iturin, surfactin, and fengycin—were identified by Liquid Chromatography Mass Spectrometry (LC-MS) (Figure 4B–D). The iturin family LPs comprised homologues with [M + 2H]+ peaks at 1058.67, 1072.66, and 1086.70 with fatty acid chains of C15, C16, and C17 lengths, respectively (Figure 4B). The surfactin family LPs comprised homologues with [M + H]+ peaks at 994.64, 1008.66, 1022.68, 1036.69, and 1050.70 with fatty acid chains of C12, C13, C14, C15, and C16 lengths, respectively (Figure 4C). The fengycin family LPs comprised homologues with [M + H]+ peaks at 1435.76, 1449.77, 1463.79, 1477.80, and 1497.82 with fatty acid chains of C14, C15, C16, C17, and C18 lengths, respectively (Figure 4D).
The AMP was purified from the VC3 fermentation broth with the concentration of ~250 mg/mL at 24 h (Figure 4E). There was no increase in AMP production after incubation up to 48 h and 72 h. The AMP sequence was identified using LC-MS/MS as a detector. The identified protein sequence matches the amino acid sequence of the protein encoded by the AMP gene (47 aa, 5.46 KDa) of VC3 and is consistent with the AMP LCI sequence of B. subtilis. The SDS-PAGE showed that the molecular weight of AMP was as expected and had high purity (Figure 4F). Prediction of the AMP protein structure using homology modeling showed that it consists of four β-folds (Figure 4G).

3.7. Effect on Spore Germination and Appressorium Formation

Conidia play important roles during M. oryzae and C. gloeosporioides infection, and therefore, the effect of VC3 free-cell filtrates on the spore germination and appressorium formation of both fungal pathogens was investigated. It was observed that, treated with 50 × VC3 free-cell filtrates, the spore germination rate of M. oryzae was significantly lower. Compared with the control (60.8%), the rate of appressorium formation was 10.7 and 29.4% after being treated with 20× or 50× VC3 free-cell filtrates (Figure 5A). Similar results were obtained in C. gloeosporioides. The 20× or 50× VC3 free-cell filtrate treatments saw reduces of 11.5% or 5.0% in spore germination, and only 10.6% or 36.7% of the germ tubes formed appressorium (Figure 5B,C). The results indicated that VC3 free-cell filtrates displayed highly efficacy in reducing the appressorium formation of M. oryzae and C. gloeosporioides.
Hardly any appressoria formation (1.2%) of M. oryzae was observed in 30 μg/mL AMP treatment. The percentage of conidia-forming appressoria in the LP treatments (14.9%) was significantly reduced compared with that of CK (65.5%), which was significantly higher than that of the AMP treatment (Figure 6A). Only 30 μg/mL AMPs inhibited the spore germination of C. gloeosporioides. A total of 30 μg/mL of AMPs and LPs resulted in significantly higher inhibition rates of appressoria formation than lower-concentration treatments, while 30 μg/mL of AMPs was the most effective in reducing the appressoria formation of C. gloeosporioides (Figure 6B). Regardless of the pathogen, AMPs displayed higher efficacy in inhibiting appressoria formation than LPs.

3.8. In Planta Biological Control

To further test the effect of B. velezensis VC3 on the antagonism against rice blast and anthracnose in peppers, the VC3 suspensions were sprayed or dripped on rice or pepper seedlings. At seven days post-inoculation, the conidia of M. oryzae produced very few lesions on the rice leaves in the VC3 treatment. In contrast, the rice seedlings in the control treatment developed numerous typical rice blast lesions (Figure 7A,B). The pepper seedlings inoculated with conidia of C. gloeosporioides developed typical water-stained lesions. Under similar conditions, conidia in the VC3 treatment failed to cause comparable disease symptoms (Figure 7C,D), and the lesion areas were significantly smaller than in the control.
Moreover, we examined the effect of AMPs and LPs on the pathogenicity of M. oryzae and C. gloeosporioides on detached barley and pepper leaves. Conidial suspensions of M. oryzae with different concentrations were drop-inoculated onto barley leaves (Figure 8A). In comparison with the control, the AMP treatment failed to cause extensive necrosis and chlorosis at 105 conidia/mL or lower, while the LP treatment failed at 104 conidia/mL or lower. Even at 105 conidia/mL, the LP treatment was less virulent (Figure 8B). On pepper leaves, which were inoculated with the conidial suspension of C. gloeosporioides, significantly smaller lesions were found with AMP treatment, and lesions produced by the LP treatments were less severe (Figure 8C,D). The statistical analysis showed that the inhibiting effect of AMP on pathogenicity was higher than that of LPs (Figure 8B,D).

4. Discussions

In the present study, the biological control potential of B. velezensis VC3 was explored. Our results highlighted a strong in vitro inhibitory activity of VC3 on mycelial growth, varying from 53.39 to 81.34% for M. oryzae, C. gloeosporioides, and S. sclerotiorum. A relatively low level of inhibition rates was seen against P. capsici, E. mrcicum, R. solani, S. rolfsii, and F. oxysporum, ranging from 32.28 to 42.02%. B. velezensis VC3 had different in vitro inhibition rates against eight fungal phytopathogens, which may be due to their nature and the modes of action involved. Pathogens possess diverse mechanisms of resistance and susceptibility, which directly impacts their responsiveness to biocontrol agents. For instance, variations in cell wall composition, enzymatic machinery, or genetic makeup among pathogens can significantly influence their susceptibility to antimicrobial agents [25]. Zhou et al. [26] proved that B. velezensis BR-01 had a strong inhibitory effect on M. oryzae, Ustilaginoidea virens, and Fusarium fujikuroi, while showed no antagonistic ability against Rhizoctonia solani. The varying inhibitory effects of B. velezensis VC3 on a range of phytopathogenic fungi underscore the complexity of biocontrol interactions. Further elucidation of the underlying mechanisms of these interactions is crucial for a comprehensive understanding of VC3’s biocontrol potential. B. velezensis VC3 exhibits a broad-spectrum antifungal activity against various phytopathogenic fungi. This aligns with the concept of developing biocontrol agents that are effective against a range of pathogens, which is crucial for sustainable agriculture and reduces the reliance on chemical fungicides.
M. oryzae and C. gloeosporioides are pathogens of both economical and scientific importance. In addition, they have arisen as model fungal pathogens for understanding the molecular basis of plant–fungus interactions [27,28]. These two pathogens tested in vitro had similar susceptibility to B. velezensis VC3, so we chose them for further investigations of their biological mechanisms.
Genomic and transcriptomic analyses revealed that B. velezensis VC3 possesses several functional gene clusters encoding the synthesis of polyketide compounds, dipeptide compounds, and other secondary metabolites. These gene clusters included the production of AMPs, LPs, and other bioactive compounds. The relative expression of the AMP synthesis gene cluster was the highest among all, being 2–4 orders of magnitude greater than those of other compound synthesis gene clusters. A previous study showed that volatile compounds from Bacillus spp. could consistently suppressed M. oryzae in vitro and in vivo [29]. Bacillomycin D isolated from B. velezensis HN-2 showed potent activity against C. gloeosporioides (Penz.) [30], and cyclic tetrapeptide from B. velezensis CE 100 displayed strong antifungal activity against C. gloeosporioides mycelial growth and spore germination [31]. This suggests that high antagonistic components are different from diverse Bacillus species isolates. Future studies are needed to investigate the antifungal roles and mechanisms of other secondary metabolites encoded by VC3.
To cause infection, the conidia attach to host leaves and produce germ tubes that can form dome-shaped infection cells called appressoria [17]. Like most fungal pathogens, spore germination and appressorium development are key steps in the colonization of host plants by M. oryzae and C. gloeosporioides. The cell-free filtrates of VC3 significantly reduced the spore germination of M. oryzae and C. gloeosporioides; however, only 30 μg/mL of AMPs inhibited the spore germination of C. gloeosporioides. It is possible that there are other secondary antimicrobial metabolites involved in spore germination inhibition, indicating that there are multiple inhibition mechanisms of VC3 on the spore germination of pathogenic fungi. The appressorium development was also significantly reduced in VC3 cell-free filtrates or active constituent treatment. The inhibition rates of cell-free filtrates or active constituents are correlated with their concentration: the higher the final concentration the stronger the inhibition. It is reported that the crude enzyme from B. velezensis CE100 inhibited the spore germination of C. gloeosporioides, causing walnut anthracnose [32]. Suspensions of B. velezensis BS1 inhibited the appressorium formation of C. scovillei [33]. The crude extract of the fermentation broth of B. velezensis ZW10 could delay the germination of M. oryzae conidia and inhibit the formation of appressorium [34]. However, the appressorium inhibition activity of AMPs and LPs against M. oryzae and C. gloeosporioides in our study has not been previously reported. Moreover, AMP induced a higher appressorium inhibition than LPs. We hypothesized that AMP may be a major factor involved in the antagonistic activities of VC3 against appressorium development.
M. oryzae and C. gloeosporioides are economically important pathogens. An inhibitory effect of VC3 suspensions was observed in M. oryzae and C. gloeosporioides infection. The inhibit rate was 84% against M. oryzae and 74% against C. gloeosporioides (Figure 7). B. subtilis isolate G5 displayed a notable biocontrol effect of 66.81% against M. oryzae in a greenhouse [6]. A previous study showed that a sterilized culture filtrate of B. subtilis DL76 had a strong ability to suppress the pathogenicity of M. oryzae, and the leaf lesion diameter was 63.5% smaller compared with the control [35]. Preventive treatment using a cell-free supernatant of Bacillus mycoides A1 diminished the severity of C. gloeosporioides by 41.9% on avocado fruit [36]. The findings of this study demonstrate that strain VC3 exhibits the potent antagonistic activity against M. oryzae and C. gloeosporioides, comparable to other strains of Bacillus spp. Additionally, strain VC3 displays a broad-spectrum antifungal effect against these two pathogens, a characteristic that has rarely been reported. This discovery not only underscores the potential of VC3 as a biocontrol agent but also highlights the need for further research to understand the unique mechanisms that confer its broad-spectrum efficacy.
The results of the pathogenicity assay on barley and pepper leaves also showed a significant effect of AMPs and LPs on the development of lesion areas caused by M. oryzae and C. gloeosporioides. Among them, AMPs can inhibit M. oryzae by up to 96% and C. gloeosporioides by up to 95% (Figure 8). It is known that appressorium-mediated penetration is required for full virulence of these two pathogens [37,38]. The inhibiting effect of AMP on pathogenicity was higher than LPs, which is consistent with the inhibiting effect on appressorium formation. Therefore, we can infer that the active constituents of B. velezensis VC3, AMP, and LPs affect the normal formation of appressoria, leading to defects in the pathogenicity of M. oryzae and C. gloeosporioides. In vitro and in planta assays further showed that AMP was more active against fungi than LPs, providing further support to the hypothesis that AMPs may serve as the main antifungal metabolite of VC3.
The mechanism of AMPs against bacteria may be attributed to the inhibition of peptidoglycan biosynthesis or degradation of peptidoglycan [39]. LPs readily bind to the bacterial surface bilayer and alter the local lipid organizational linkages on negatively charged fatty acids, ultimately restructuring the lipid bilayer and thus preventing cellular processes [40]. While the antimicrobial effects of AMPs and LPs against bacteria are established, its action and mechanism against pathogenic fungi need to be analyzed in depth. In this study, we showed that AMPs and LPs directly inhibited appressorium formation and the invasion of M. oryzae and C. gloeosporioides in plant tissues. The results expand the antimicrobial spectrum of AMPs and LPs and the potential for their application in the biological control of pathogenic fungi.

5. Conclusions

In conclusion, the present study emphasizes the potential of B. velezensis strain VC3 as a biocontrol agent. The broad-spectrum inhibitory effect of VC3 on various phytopathogenic fungi, especially M. oryzae and C. gloeosporioides, was demonstrated through in vitro and in planta experiments. Moreover, VC3 contains several functional gene clusters responsible for the synthesis of antifungal secondary metabolites, including AMPs and LPs. The most effective actions of VC3 include its multifaceted approach to inhibiting appressorium development, which is a critical step in the pathogenic process of these fungi. The potential applications of B. velezensis VC3 in agriculture are vast, particularly in the development of biopesticides. These biopesticides could offer a more environmentally friendly alternative to chemical pesticides and could mitigate the risk of developing resistance in M. oryzae and C. gloeosporioides.
However, to fully harness the potential of VC3 in agricultural practices, understanding the molecular mechanisms by which VC3 exerts its biocontrol effects is essential. This knowledge can inform the development of genetically enhanced strains with improved biocontrol properties or the discovery of novel bioactive compounds that can be synthesized for use in biopesticide formulations. In addition, future research should explore the effectiveness of VC3 under different environmental conditions and evaluate the long-term biocontrol activity of VC3.
In summary, B. velezensis VC3 shows great promise as a biocontrol agent against M. oryzae and C. gloeosporioides, with potential applications in agriculture. However, continued research is necessary to maximize its benefits and ensure its effective integration into farming practices.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pr12071490/s1, Table S1: Effect of B. velezensis VC3 on growth of rice and pepper; Table S2: The information of VC3 genome and its comparison with B. velezensis FZB42 and QST713 strains; Table S3: Analysis of relative gene expression levels in VC3.

Author Contributions

Conceptualization, X.T.; methodology, K.L., X.Z., Y.C., T.Z. and Q.S.; investigation, K.L., X.Z., Y.C., T.Z., Q.S. and H.W.; writing—original draft, K.L. and X.Z.; writing—review and editing, Z.C. and X.T.; visualization, K.L. and X.Z.; project administration, Y.S. and X.T.; funding acquisition, D.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Key Research and Development Program of China (2021YFE0111200), the Tsinghua University Initiative Scientific Research Program (20223080016), and the Key Research and Development Program of Hunan Province (2022NK2050, 2023NK2014).

Data Availability Statement

The data are contained within the article and Supplementary Materials.

Conflicts of Interest

Authors Hongtao Wu and Yan Sun are employed by Jiangsu Tsingda Smart Biotech Co., Ltd. Xin Zhang and Xinqiu Tan are employed by the Yuelushan Laboratory. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Inhibitory effects of B. velezensis VC3 against eight fungal phytopathogens. CK: pathogenic fungi with LB + glucose medium on PDA medium; VC3: pathogenic fungi with B. velezensis VC3 on PDA medium. The plates were incubated in a 28 °C incubator and cultured for four to seven days. (A) M. oryzae; (B) C. gloeosporioides; (C) S. sclerotiorum; (D) P. capsici; (E) E. mrcicum; (F) F. oxysporum; (G) R. solani; (H) S. rolfsii.
Figure 1. Inhibitory effects of B. velezensis VC3 against eight fungal phytopathogens. CK: pathogenic fungi with LB + glucose medium on PDA medium; VC3: pathogenic fungi with B. velezensis VC3 on PDA medium. The plates were incubated in a 28 °C incubator and cultured for four to seven days. (A) M. oryzae; (B) C. gloeosporioides; (C) S. sclerotiorum; (D) P. capsici; (E) E. mrcicum; (F) F. oxysporum; (G) R. solani; (H) S. rolfsii.
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Figure 2. Evolutionary relationships, genome annotation, and gene classification of VC3. (A) Phylogenetic neighbor-joining tree reconstructed based on 16S rRNA, atpD, gyrA, gyrB, recN, topA, uvrC, ftsZ, and infB genes of the selected strains of bacteria; (B) NR homologous species distribution of VC3 genome; (C) KEGG functional classification of VC3 genome; (D) CAZy gene classification in the VC3 genome; (E) COG functional classification of VC3 genome.
Figure 2. Evolutionary relationships, genome annotation, and gene classification of VC3. (A) Phylogenetic neighbor-joining tree reconstructed based on 16S rRNA, atpD, gyrA, gyrB, recN, topA, uvrC, ftsZ, and infB genes of the selected strains of bacteria; (B) NR homologous species distribution of VC3 genome; (C) KEGG functional classification of VC3 genome; (D) CAZy gene classification in the VC3 genome; (E) COG functional classification of VC3 genome.
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Figure 3. Relative expression levels of biocontrol-related secondary metabolite gene clusters. (A) AMP; (B) surfactin; (C) iturin; (D) fengycin; (E) difficidin; (F) bacillaene; (G) macrolactin H; (H) bacillibactin; (I) bacilysin. Note: Blue and red bars represent VC3 gene expression levels after 24 h and 48 h of incubation, respectively.
Figure 3. Relative expression levels of biocontrol-related secondary metabolite gene clusters. (A) AMP; (B) surfactin; (C) iturin; (D) fengycin; (E) difficidin; (F) bacillaene; (G) macrolactin H; (H) bacillibactin; (I) bacilysin. Note: Blue and red bars represent VC3 gene expression levels after 24 h and 48 h of incubation, respectively.
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Figure 4. Separation, purification, and characterization of LPs and AMP. (A) Content of LPs in broth; (B) molecular ion peaks of three iturin monomers; (C) molecular ion peaks of five surfactin monomers; (D) molecular ion peaks of five fengycin monomers; (E) content of AMP in broth; (F) SDS-PAGE of AMP; (G) protein structure of AMP.
Figure 4. Separation, purification, and characterization of LPs and AMP. (A) Content of LPs in broth; (B) molecular ion peaks of three iturin monomers; (C) molecular ion peaks of five surfactin monomers; (D) molecular ion peaks of five fengycin monomers; (E) content of AMP in broth; (F) SDS-PAGE of AMP; (G) protein structure of AMP.
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Figure 5. VC3 cell-free filtrates inhibit the conidia germination and appressorium formation of M. oryzae (A) and C. gloeosporioides (B). The error bars represent the standard errors. The asterisks (*) represent significant differences compared with CK according to the multiple Duncan test (p < 0.05). Corresponding ANOVA results: conidia germination and appressorium formation of M. oryzae, F2,6 = 3.551, p = 0.096, F2,6 = 49.412, p < 0.001; C. gloeosporioides, F2,6 = 76.652, p < 0.001, F2,6 = 103.536, p < 0.001. (C) Morphological observations of conidia. Bar = 10 μm. CK: control, treated with LB + glucose medium; 20×: treated with VC3 cell-free filtrates at a concentration of 5%; 50×: treated with VC3 cell-free filtrates at a concentration of 2%.
Figure 5. VC3 cell-free filtrates inhibit the conidia germination and appressorium formation of M. oryzae (A) and C. gloeosporioides (B). The error bars represent the standard errors. The asterisks (*) represent significant differences compared with CK according to the multiple Duncan test (p < 0.05). Corresponding ANOVA results: conidia germination and appressorium formation of M. oryzae, F2,6 = 3.551, p = 0.096, F2,6 = 49.412, p < 0.001; C. gloeosporioides, F2,6 = 76.652, p < 0.001, F2,6 = 103.536, p < 0.001. (C) Morphological observations of conidia. Bar = 10 μm. CK: control, treated with LB + glucose medium; 20×: treated with VC3 cell-free filtrates at a concentration of 5%; 50×: treated with VC3 cell-free filtrates at a concentration of 2%.
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Figure 6. Effect of AMPs and LPs on inhibiting conidia germination and appressorium formation of M. oryzae (A) and C. gloeosporioides (B). CK: control, treated with sterile distilled water; AMPs or LPs: treated with AMPs or LPs at a final concentration of 30 μg/mL or 6 μg/mL. The error bars represent the standard errors. The asterisks (*) represent significant differences compared with CK according to the multiple Duncan test (p < 0.05). Corresponding ANOVA results: 2 hpi or 4 hpi conidia germination and appressorium formation of M. oryzae, F4,10 = 2.218, p = 0.140, F4,10 = 6.865, p = 0.006, F4,10 = 97.128, p < 0.001; C. gloeosporioides, F4,10 = 4.103, p = 0.032, F4,10 = 2.016, p = 0.168, F4,10 = 151.914, p < 0.001.
Figure 6. Effect of AMPs and LPs on inhibiting conidia germination and appressorium formation of M. oryzae (A) and C. gloeosporioides (B). CK: control, treated with sterile distilled water; AMPs or LPs: treated with AMPs or LPs at a final concentration of 30 μg/mL or 6 μg/mL. The error bars represent the standard errors. The asterisks (*) represent significant differences compared with CK according to the multiple Duncan test (p < 0.05). Corresponding ANOVA results: 2 hpi or 4 hpi conidia germination and appressorium formation of M. oryzae, F4,10 = 2.218, p = 0.140, F4,10 = 6.865, p = 0.006, F4,10 = 97.128, p < 0.001; C. gloeosporioides, F4,10 = 4.103, p = 0.032, F4,10 = 2.016, p = 0.168, F4,10 = 151.914, p < 0.001.
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Figure 7. Pathogenicity assay under VC3 suspensions treatments. (A) Rice seedlings were inoculated with the conidia of M. oryzae with or without VC3 suspensions. Graph (B) shows the mean lesion density. (C) Pepper leaves were inoculated with the conidia of C. gloeosporioides with or without VC3 suspensions. Graph (D) shows the size of the lesion area. CK: control, treated with LB + glucose medium; VC3: treated with VC3 cell-free filtrates at a concentration of 10%. The error bars represent the standard errors. Asterisks above the bars indicate significant differences compared with CK according to the multiple Duncan test (p < 0.05). Corresponding ANOVA results: rice, F1,4 = 2547.368, p < 0.001; pepper, F1,10 = 57.917, p < 0.001.
Figure 7. Pathogenicity assay under VC3 suspensions treatments. (A) Rice seedlings were inoculated with the conidia of M. oryzae with or without VC3 suspensions. Graph (B) shows the mean lesion density. (C) Pepper leaves were inoculated with the conidia of C. gloeosporioides with or without VC3 suspensions. Graph (D) shows the size of the lesion area. CK: control, treated with LB + glucose medium; VC3: treated with VC3 cell-free filtrates at a concentration of 10%. The error bars represent the standard errors. Asterisks above the bars indicate significant differences compared with CK according to the multiple Duncan test (p < 0.05). Corresponding ANOVA results: rice, F1,4 = 2547.368, p < 0.001; pepper, F1,10 = 57.917, p < 0.001.
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Figure 8. Pathogenicity assay under active constituent treatments. (A) Barley seedlings were drop-inoculated with the conidia of M. oryzae (0.2% gelatin) with or without active constituents. The concentration of each conidia suspension is marked on the right. Graph (B) shows the lesion size on rice leaves. Inoculation with 0.2% gelatin was used as the control (CK). Corresponding ANOVA results: 105, F2,9 = 40.828, p < 0.001; 104, F2,9 = 24.684, p < 0.001; 103, F2,9 = 4.383, p = 0.047. (C) Pepper leaves were inoculated with the conidia of C. gloeosporioides with or without VC3 suspensions. The conidial suspensions were used as controls (CK). Graph (D) shows the size of the lesion area on pepper leaves. AMPs or LPs: treated with AMPs or LPs at a final concentration of 30 μg/mL. Corresponding ANOVA results: F2,9 = 74.272, p < 0.001. Error bars represent the standard errors. Asterisks above the bars indicate significant differences compared with CK according to the multiple Duncan test (p < 0.05).
Figure 8. Pathogenicity assay under active constituent treatments. (A) Barley seedlings were drop-inoculated with the conidia of M. oryzae (0.2% gelatin) with or without active constituents. The concentration of each conidia suspension is marked on the right. Graph (B) shows the lesion size on rice leaves. Inoculation with 0.2% gelatin was used as the control (CK). Corresponding ANOVA results: 105, F2,9 = 40.828, p < 0.001; 104, F2,9 = 24.684, p < 0.001; 103, F2,9 = 4.383, p = 0.047. (C) Pepper leaves were inoculated with the conidia of C. gloeosporioides with or without VC3 suspensions. The conidial suspensions were used as controls (CK). Graph (D) shows the size of the lesion area on pepper leaves. AMPs or LPs: treated with AMPs or LPs at a final concentration of 30 μg/mL. Corresponding ANOVA results: F2,9 = 74.272, p < 0.001. Error bars represent the standard errors. Asterisks above the bars indicate significant differences compared with CK according to the multiple Duncan test (p < 0.05).
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Table 1. Effect of fungal antagonists on the in vitro mycelial growth of B. velezensis VC3.
Table 1. Effect of fungal antagonists on the in vitro mycelial growth of B. velezensis VC3.
TreatmentsInhibition Rate (%)
Magnaporthe oryzae59.28 ± 1.18 b
Colletotrichum gloeosporioides53.39 ± 6.59 b
Sclerotinia sclerotiorum81.34 ± 5.34 a
Phytophthora capsici34.96 ± 4.04 c
Exserohilum mrcicum32.28 ± 13.92 c
Fusarium oxysporum40.53 ± 1.64 c
Rhizoctonia solani35.86 ± 8.69 c
Selerotium rolfsii42.02 ± 7.58 c
Note: Each value of the inhibition rates is the mean (±SE) of at least three replications. Values in columns followed by the same letters indicate no significant difference according to the multiple Duncan test (p < 0.05). Corresponding ANOVA results: F7,29 = 23.083, p < 0.001.
Table 2. Gene clusters involved in biocontrol.
Table 2. Gene clusters involved in biocontrol.
ProductGene ClusterSize (bp)
Fengycin (NRPS a)fenABCDE47,585
Surfactin (NRPS)srfAABCD43,672
Iturin (NRPS)ituDABC69,391
Bacillibactin (PKS b)dhbACEBF51,791
Bacillaene (PKS)baeBCDE, acpK, baeGHIJLMNRS72,467
Difficidin (transAT-PKS)difABCDEFGHIJKLMNO70,775
Macrolactin H (transAT-PKS)mlnABCDEFGHI54,820
Bacilysin (dipeptide)bacABCDEFG41,419
AntipeptideAMP LCI285
a NRPS: non-ribosomal peptide synthetases; b PKS: polyketide synthases.
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MDPI and ACS Style

Li, K.; Zhang, X.; Chen, Y.; Zhang, T.; Sun, Q.; Wu, H.; Chen, Z.; Sun, Y.; Liu, D.; Tan, X. A Promising Biocontrol Agent of Bacillus velezensis VC3 against Magnaporthe oryzae and Colletotrichum gloeosporioides in Plants. Processes 2024, 12, 1490. https://doi.org/10.3390/pr12071490

AMA Style

Li K, Zhang X, Chen Y, Zhang T, Sun Q, Wu H, Chen Z, Sun Y, Liu D, Tan X. A Promising Biocontrol Agent of Bacillus velezensis VC3 against Magnaporthe oryzae and Colletotrichum gloeosporioides in Plants. Processes. 2024; 12(7):1490. https://doi.org/10.3390/pr12071490

Chicago/Turabian Style

Li, Kui, Xin Zhang, Yue Chen, Tong Zhang, Qianlong Sun, Hongtao Wu, Zhen Chen, Yan Sun, Dehua Liu, and Xinqiu Tan. 2024. "A Promising Biocontrol Agent of Bacillus velezensis VC3 against Magnaporthe oryzae and Colletotrichum gloeosporioides in Plants" Processes 12, no. 7: 1490. https://doi.org/10.3390/pr12071490

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