From Strain Characterization to Field Authorization: Highlights on Bacillus velezensis Strain B25 Beneficial Properties for Plants and Its Activities on Phytopathogenic Fungi
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacillus velezensis Strain B25 Genomic Features Assessment
2.1.1. Genome Characterization
2.1.2. Gene Analysis
2.2. Assessment of the Inhibition of Fungal Pathogens by Bacillus velezensis Strain B25
2.2.1. B25 Culture Conditions
2.2.2. In Vitro Confrontations for Antagonism Potential Evaluation
2.2.3. Bioassay in Growth Chamber for Biocontrol Evaluation against F. graminearum Strain B377
2.3. Bacillus velezensis Strain B25 Metabolic Properties Assessment
2.3.1. Phosphate Solubilization Assay
2.3.2. Siderophores Detection Assay
2.3.3. Indolacetic Acid (IAA) Production Assay
2.3.4. Metabolic Profiling on Biolog GenIII Microplates
2.3.5. Antibiograms and Minimal Inhibitory Concentrations Evaluation
2.3.6. Growth Response under UV-C Stress
2.4. Bacillus velezensis Strain B25 Ecotoxicological Impact on Representative Organisms
2.4.1. Effects on Mortality and Reproduction of Eisenia fetida
2.4.2. Chronic Toxicity Effect on Daphnia magna
2.5. Statistical Analysis
3. Results
3.1. Characterization of the General Genomic Features of Strain B25
3.1.1. Genome Specificities
3.1.2. Phylogenetic Relationship of Bacillus velezensis Strain B25
3.1.3. Genes Involved in Biocontrol
3.1.4. Detection of Resistance Genes to Antibiotics
3.1.5. Polysaccharide Degrading Enzymes
3.2. Characterization of the Biocontrol Properties of Bacillus velezensis Strain B25
3.2.1. In Vitro Microbial Interactions Tests
- (i)
- A fast increase of the II was observed for the B25 challenge co-cultures against B278, B375, B376, B377, B381, B383, and B385 (Figure 3); with a 2-day latency phase followed by an exponential-like increase in 7 days, and finally a plateau with II reaching 0.7532, 0.8970, 0.8107, 0.6850, 0.6010, 0.6898, and 0.7243, respectively. For B381 strain, the plateau was not the maximum II reached (0.9626 at 7 days of culture followed by a decrease to the stationary phase). It could be explained by a strong antibiosis of B25 until 7 days before another phase of pathogen growth, however insufficient to resist B25.
- (ii)
- A longer latency phase (4 days) followed by a gradual increase of the II until the end of the experiment (15 days) was observed for the B25 challenge co-cultures against B378, B379, B380, B382, B384, and B386, with maximum II reaching 0.4052, 0.4516, 0.4663, 0.6010, 0.6854, and 0.8157, respectively (Figure 4).
3.2.2. In Planta Biocontrol Activities
3.3. Characterization of the Metabolic Properties of Strain B25
3.3.1. Phosphate Solubilization
3.3.2. Siderophores Detection
3.3.3. IAA Production
3.3.4. Metabolic Profiling on Biolog GenIII Microplates
3.3.5. Minimal Inhibitory Concentrations (MICs) of Antibiotics
3.3.6. UV Sensitivity
3.4. Characterization of the Ecotoxicological Impact of Strain B25
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
Ethical Approval
References
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Bacillus velezensis B25 | Bacillus methylotrophicus FZB42 | Bacillus amyloliquefaciens IT-45 | Bacillus subtilis 168 | Bacillus licheniformis ATCC14580 | |
---|---|---|---|---|---|
Genome size (bp) | 3,854,619 | 3,918,589 | 3,928,857 | 4,214,630 | 4,222,336 |
G+C content (%) | 46.71 | 46.4 | 46.62 | 43.5 | 46.2 |
Protein-coding sequences | 3683 | 3693 | 3949 | 4106 | 4208 |
Average CDS size (bp) | 934 | 895 | 888 | 896 | 873 |
Percentage of coding region | 89 | 88 | 89 | 87 | 86 |
Ribosomal RNA operons | 7 | 10 | 10 | 10 | 7 |
Number of tRNAs | 68 | 89 | 95 | 86 | 72 |
Reference study and GenBank accession number | Gerbore et al. [16] LN999829.1 | Chen et al. [33] CP000560.2 | Tompkins et al. [34] CP004065.1 | Kunst et al. [35] AL009126.3 | Rey et al. [36] CP000002.3 |
Compound | Enzyme | Gene | High Similarity with | Reference Study |
---|---|---|---|---|
Surfactin | NRPS | srfABCD | FZB42 | Chen et al. [33] |
Fengycin | NRPS | fenABCDE | FZB42 | |
Bacillibactin | NRPS | dhbABCDEF | FZB42 | |
Bacilysin | NRPS | bacABCDEFG | FZB42 | |
Macrolactin | PKS | minABCDEFGHI | FZB42 | |
Bacillaene | PKS/NRPS | baeBCDE, acpK, baeGHIJLMNRS | FZB42 | |
Difficidin | PKS | dfnAYXBCDEFGHIJKLM | FZB42 | |
Mycosubtilin | NRPS | mycABC | IT-45 | Tompkins et al. [34] |
Amylolysin | RP | amyIEKRAMT | GA1 | Arguelles-Arias [37] |
B25 Gene Label | Gene Symbol | Product | Antibiotic Class | Similarity with (Amino Acid Identity %) |
---|---|---|---|---|
BAMMD1_0254 | lmrB | Lincomycin resistance protein | lincosamide | IT45 (99.8%), FZB42 (99.4%), B168 (88.4%) |
BAMMD1_0286 | Mdr | multidrug-efflux transporter | aminonucleoside, fluoroquinolone | IT45 (100%), FZB42 (99.6%), B168 (90.2%) |
BAMMD1_0294 | tmrB | Tunicamycin resistance protein | nucleoside | IT45 (98.5%), FZB42 (98.5%), B168 (77%) |
BAMMD1_0514 | Cfr | Ribosomal RNA large subunit methyltransferase | oxazolidinone, lincosamide, streptogramin, phenicol | IT45 (99.7%), FZB42 (97.4%) |
BAMMD1_0534 | vmlR | Ribosome protection protein | streptogramin, oxazolidinone, lincosamide, pleuromutilin, phenicol, tetracycline, macrolide | IT45 (100%), FZB42 (98.3%), B168 (71%) |
BAMMD1_0665 | aadK | Aminoglycoside 6-adenylyltransferase | aminoglycoside | IT45 (99.3%), B168 (63%) |
BAMMD1_1112 | Bla | Metallo-beta-lactamase type 2 | Beta-lactam | IT45 (99.6%), FZB42 (97.2%) |
BAMMD1_1128 | penN | Beta-lactamase | Beta-lactam | IT45 (99.7%), FZB42 (95.7%), B168 (88.5%) |
BAMMD1_2341 | tetB | Tetracycline resistance protein | tetracycline | IT45 (99.3%), B168 (86.7%) |
Treatment | FSB Disease Severity | |
---|---|---|
Test 1 | Test 2 | |
Control | 0.9 (± 0.31) ab | 0 (± 0) a |
Bacillus velezensis B25 | 0 (± 0) a | 0.12 (± 0.12) a |
Fusarium graminearum B377 | 6.5 (± 1.33) c | 8.38 (± 1.51) b |
B25 + B377 | 4.41(± 1.1) bc | 4.59 (± 0.96) c |
Substrates of Biolog Gen III | |||||
---|---|---|---|---|---|
Negative Control | − | D-Lactic Acid Methyl Ester | + | l-Aspartic Acid | + |
Dextrin | + | l-Lactic Acid | + | l-Glutamic Acid | + |
d-Maltose | + | Citric Acid | + | l-Histidine | + |
d-trehalose | + | α-Keto-Glutaric Acid | + | l-Pyroglutamic Acid | + |
d-Cellobiose | + | d-Malic Acid | + | l-Serine | + |
Gentiobiose | + | l-Malic Acid | + | Pectin | + |
Sucrose | + | Bromo-Succinic Acid | + | d-Galacturonic Acid | + |
d-Turanose | + | Tween 40 | + | l-Galactonic Acid Lactone | + |
Stachyose | + | ϒ-Amino-Butyric Acid | + | Positive control | + |
d-Raffinose | + | α-Hydroxy-Butyric Acid | − | pH 6 | + |
α-d-Lactose | + | β-Hydroxy-d, l-Butyric Acid | + | pH 5 | + |
d-Melibiose | + | α-Keto-Butyric Acid | − | 1% NaCl | + |
β-Methyl-d-Glucoside | + | Acetoacetic Acid | + | 4% NaCl | + |
d-Salicin | + | Propionic Acid | + | 8% NaCl | + |
N-Acetyl-d-Glucosamine | + | Acetic Acid | + | 1% Sodium Lactate | + |
N-Acetyl-d-Mannosamine | + | Formic Acid | + | Fusidic acid | − |
N-Acetyl-d-Galactosamine | + | l-Fucose | + | d-Serine-2 | − |
N-Acetyl Neuraminic Acid | + | l-Rhamnose | + | Troleandomycin | − |
α-d-Glucose | + | Inosine | + | Rifamycin SV | − |
d-Mannose | + | d-Sorbitol | + | Minocycline | − |
d-Fructose | + | d-Mannitol | + | Lincomycin | + |
d-Galactose | + | d-Arabitol | + | Guanidine HCl | + |
3-Methyl Glucose | + | Myo-inositol | + | Niaproof 4 | − |
d-Fucose | + | Glycerol | + | Vancomycin | − |
d-Gluconic Acid | + | d-Glucose-6-PO4 | + | Tetrazolium violet | + |
d-Glucuronic Acid | + | d-Fructose-6-PO4 | + | Tetrazolium Blue | − |
Glucuronamide | + | d-Aspartic Acid | + | Nalidixic Acid | − |
Music Acid | + | d-Serine | − | Lithium Chloride | + |
Quinic Acid | + | Gelatin | + | Potassium Tellurite | + |
d-Saccharic Acid | + | Glycyl-l-Proline | + | Aztreonam | − |
p-Hydroxy-Phenylacetic Acid | − | l-Alanine | + | Sodium Butyrate | + |
Methyl pyruvate | + | l-Arginine | + | Sodium Bromate | − |
Antibiotics | Imipenem | Amikacin | Ceftazidime | Ciprofloxacin | Vancomycin | Erythromycin | Tetracycline |
---|---|---|---|---|---|---|---|
standard range [µg·mL−1] | [32–0.002] | [256–0.015] | [256–0.015] | [32–0.002] | [256–0.015] | [256–0.015] | [256–0.015] |
MIC for Bacillus B25 [µg·mL−1] | 0.015 | 1 | 32 | 0.03 | 0.5 | 0.12 | 4 |
Exposure Time under UV-C (Minutes) | |||||||
---|---|---|---|---|---|---|---|
Bacterial strains | 0 | 5 | 10 | 15 | 20 | 25 | 30 |
Bacillus B25 | + | + | + | − | − | − | − |
Bacillus IT45 | + | − | − | − | − | − | − |
Product Tested | Bacillus velezensis Strain B25 (1 × 108 cfu·g−1) | |||
---|---|---|---|---|
Assay Element/Substrate | Eisenia fetida/Artificial Soil | |||
Application Rates (kg·ha−1) | Average Mortality at 28 Days (%) * | Biomass Increase (Average %) ** | Fecundity | |
Average Number per Adult | Effects (%) *** | |||
Control | 0 | +80.69 | 17.84 | NA |
1 | 1.30 (NS) | +87.20 (NS) | 16.93 (NS) | −5.63 (NS) |
2 | −3.90 (NS) | +86.84 (NS) | 14.33 (NS) | −20.14 (NS) |
10 | −3.90 (NS) | +85.51 (NS) | 17.15 (NS) | −4.39 (NS) |
NOEC (kg·ha−1) | 10 | |||
EC50 (kg·ha−1) | >10 |
Endpoint | Concentration (mg·L−1) | |
---|---|---|
Immobilization | EC10 (95% confidence limits) | 2853 (Not determined) |
Immobilization | EC20 (95% confidence limits) | 4484 (Not determined) |
Immobilization | EC50 (95% confidence limits) | 9712 (Not determined) |
No Observed Effect Concentration | 4500 | |
Lowest Observed Effect Concentration | 10,000 | |
Body length | EC10 (95% confidence limits) | 5963 (4413 to 8058) |
Body length | EC20 (95% confidence limits) | >10,000 (Not determined) |
Body length | EC50 (95% confidence limits) | >10,000 (Not determined) |
No Observed Effect Concentration | 2200 | |
Lowest Observed Effect Concentration | 4500 | |
Reproduction | EC10 (95% confidence limits) | 4240 (Not determined) |
Reproduction | EC20 (95% confidence limits) | 5062 (Not determined) |
Reproduction | EC50 (95% confidence limits) | 6855 (Not determined) |
No Observed Effect Concentration | 4500 | |
Lowest Observed Effect Concentration | 10,000 |
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Joly, P.; Calteau, A.; Wauquier, A.; Dumas, R.; Beuvin, M.; Vallenet, D.; Crovadore, J.; Cochard, B.; Lefort, F.; Berthon, J.-Y. From Strain Characterization to Field Authorization: Highlights on Bacillus velezensis Strain B25 Beneficial Properties for Plants and Its Activities on Phytopathogenic Fungi. Microorganisms 2021, 9, 1924. https://doi.org/10.3390/microorganisms9091924
Joly P, Calteau A, Wauquier A, Dumas R, Beuvin M, Vallenet D, Crovadore J, Cochard B, Lefort F, Berthon J-Y. From Strain Characterization to Field Authorization: Highlights on Bacillus velezensis Strain B25 Beneficial Properties for Plants and Its Activities on Phytopathogenic Fungi. Microorganisms. 2021; 9(9):1924. https://doi.org/10.3390/microorganisms9091924
Chicago/Turabian StyleJoly, Pierre, Alexandra Calteau, Aurélie Wauquier, Rémi Dumas, Mylène Beuvin, David Vallenet, Julien Crovadore, Bastien Cochard, François Lefort, and Jean-Yves Berthon. 2021. "From Strain Characterization to Field Authorization: Highlights on Bacillus velezensis Strain B25 Beneficial Properties for Plants and Its Activities on Phytopathogenic Fungi" Microorganisms 9, no. 9: 1924. https://doi.org/10.3390/microorganisms9091924