Broad-Spectrum Antifungal, Biosurfactants and Bioemulsifier Activity of Bacillus subtilis subsp. spizizenii—A Potential Biocontrol and Bioremediation Agent in Agriculture
Abstract
:1. Introduction
2. Results
2.1. Morphological, Physiological and Biochemical Characterization and Molecular Identification of Bacillus subtilis MC6B-22 Strain
2.2. Detection of Lipopeptides by PCR
2.3. Genome Sequencing and Analysis
2.4. Growth Kinetic and Production of Crude Lipopeptide from Bacillus subtilis MC6B-22 with Antifungal, Biosurfactant and Bioemulsifier Activities
2.5. Partial Purification of Crude Lipopeptide Fraction
2.6. Antifungal and Bioemulsifier Activities
2.7. Stability of the Emulsifying Activity of CL84
3. Discussion
3.1. Physiology, Biochemistry and Identification of Bacillus subtilis MC6B-22
3.2. Genetic Prospection of Lipopeptides
3.3. Growth Kinetic and Production of Crude Lipopeptide from Bacillus subtilis MC6B-22 with Antifungal, Biosurfactant and Bioemulsifier Activities
3.4. Partial Purification of Crude Lipopeptide Fraction
3.5. Antifungal and Bioemulsifier Activities
4. Materials and Methods
4.1. Morphology, Physiological and Biochemical of MC6B-22 Strain
4.2. Phylogenetic Analysis of MC6B-22 and Genetic Characterization of Lipopeptides
4.3. Genome Sequencing and Analysis
4.4. Growth Kinetic and Production of Compounds with Antifungal, Hemolytic, Biosurfactant and Bioemulsifier Activity
4.4.1. Extraction of Crude Lipopeptide
4.4.2. Antifungal Activity
4.4.3. Hemolytic Activity
4.4.4. Drop-Collapsing Test
4.4.5. Emulsifying Activity Determination
4.5. TLC Bioautography Analysis of CL84
4.6. Partial Purification and Characterization of Crude Lipopeptide Fraction
4.6.1. Reverse-Phase HPLC Analysis
4.6.2. Mass Spectrometry
4.7. Isolation of Different Phytopathogens
4.8. Fungicidal and Fungistatic Activity
4.9. Effect of Salinity, pH and Temperature on Bioemulsifier Activity
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Characteristics | |||
---|---|---|---|
Gram reaction | + | Degradation of | |
Spore shape | Oval | Carboxymethyl cellulose | − |
Growth microaerophilic | + | Use of | |
Catalase | + | Citrate | + |
Oxidase | + | Growth at | |
Motile | + | pH 5.6 | + |
Nitrate reduction | + | 50 °C | + |
Production of | Growth in presence of | ||
Indole | − | LBM | + |
H2S | − | 2% NaCl | + |
pH in Vosges-Proskauer | + | 5% NaCl | + |
Hydrolysis of | 6.5% NaCl | + | |
Casein | + | 10% NaCl | + |
Starch | + | Fermentation of | |
Urea | + | Mannitol | − |
Tween 80 | − | Glycerol | − |
Egg yolk lecithin | − | Glucose | + |
Matched Strain | Score Range | Overlap | e-Value | Identity Range | Acc. Number | 16S RNA Length | 16S RNA Copy Number |
---|---|---|---|---|---|---|---|
Bacillus subtilis strain FDAARGOS_606 | 2874 | 100% | 0 | 100% | CP041015.1 | 1556 | 10 |
Bacillus subtilis subsp. spizizenii str. W23 | 2868–2874 | 100% | 0 | 99.94–100% | CP002183.1 | 1556 | 10 |
Bacillus intestinalis strain T30 | 2868–2874 | 100% | 0 | 99.94–100% | CP011051.1 | 1556 | 10 |
N | Type | Location | Most Similar Known Cluster (Similarity 1) | Most Similar Known Cluster Type | Activity |
---|---|---|---|---|---|
1 | TransAT-PKS, PKS-like, T3PKS, NRPS | 430,447–535,671 | Bacillaene (100%) | Polyketide + NRP | Polyene antibiotic that is active against a wide range of bacteria [28]. |
2 | NRPS, betalactone, transAT-PKS | 602,362–678,373 | Mycosubtilin (100%) | NRP + Polyketide | Polypeptide with antifungal and hemolytic activities, belonging to the iturin lipopeptide family [29]. |
3 | Terpene | 795,235–816,705 | Diverse functions: infochemicals for inter and intraspecies communication, response to temperature, oxidative and osmotic stress, antimicrobial, anti-oxidative, anti-inflammatory and anti-cancer [30]. | ||
4 | T3PKS | 864,155–905,252 | 1-carbapen-2-em-3-carboxylic acid (16%) | Other | Antibiotic with activity against Gram-positive and Gram-negative bacteria [31]. |
5 | NRP-metallophore, NRPS, RiPP-like | 1,759,322–1,811,439 | Bacillibactin (100%) | NRP | Catecholate siderophore capable of binding and solubilizing iron. It chelates Fe3+ from the environment, which is later transported into the bacterial cytoplasm. Some Bacillibactins have shown bactericidal activity against multidrug-resistant strains [32]. |
6 | Lanthipeptide-class-i | 1,962,423–1,988,648 | Subtilin (100%) | RiPP:Lanthipeptide | Lantibiotic originally produced by Bacillus subtilis ATCC6633. It shows antimicrobial activity against Gram-positive bacteria [33]. |
7 | CDPS | 2,094,187–2,114,933 | Pulcherriminic acid (100%) | Other | Cyclic dipeptide that chelates Fe3+. This property allows competition for environmental iron ions to achieve bacteriostatic effects [34]. |
8 | Sactipeptide, other | 2,351,558–2,413,975 | Bacilysin (100%) | Other | Induction of bacteria and fungi cell wall lysis [35]. |
9 | Phosphonate | 2,951,523–2,971,909 | Rhizocticin A (100%) | Other | Hydrophilic phosphono-oligopeptide with antifungal activity [36]. |
10 | NRPS | 3,098,092–3,163,483 | Surfactin (86%) | NRP: Lipopeptide | Antibacterial, antifungal and antiviral agent; surfactant, antineoplastic and a platelet aggregation inhibitor; cyclodepsipeptide and a macrocyclic lactone [37]. |
11 | Terpene | 3,867,459–3,888,265 | No match | No match | Many putative terpene synthase genes have been discovered in bacteria [38]. |
Time (h) | OD520 nm | Vegetative Cells (CFU/mL) | Spores (CFU/mL) | pH | CL (mg/L) | Antifungal (mm) | Hemolyticg (mm) | Biosurfactan 1 (mm) | Emulsifying EI24 (%) |
---|---|---|---|---|---|---|---|---|---|
12 | 1.94 ± 0.20 | 2.33 × 108 | 4.86 × 105 | 7.51 ± 0.04 | 289 ± 80 | (–) | (–) | 4.0 | (–) |
24 | 3.13 ± 0.23 | 5.36 × 108 | 6.00 × 105 | 7.90 ± 0.04 | 239 ± 70 | 12 | (–) | 4.0 | 5 |
36 | 3.35 ± 0.31 | 6.54 × 108 | 5.83 × 106 | 8.36 ± 0.01 | 479 ± 10 | 19 | 9 | 4.5 | 49.52 |
48 | 5.53 ± 0.20 | 3.21 × 109 | 3.60 × 107 | 8.23 ± 0.03 | 359 ± 11 | 24 | 14 | 5.6 | 58.13 |
60 | 6.96 ± 0.58 | 3.50 × 1011 | 1.69 × 107 | 8.22 ± 0.00 | 366 ± 02 | 25 | 14 | 5.8 | 58.71 |
72 | 5.86 ± 0.42 | 1.41 × 1011 | 2.69 × 109 | 8.57 ± 0.02 | 435 ± 03 | 22 | 13 | 5.0 | 59.01 |
84 | 5.04 ± 0.15 | 7.72 × 1010 | 3.82 × 1010 | 8.49 ± 0.01 | 556 ± 79 | 27 | 15 | 6.0 | 59.03 |
96 | 5.85 ± 0.41 | 5.14 × 1010 | 1.96 × 1010 | 8.71 ± 0.02 | 481 ± 05 | 24 | 13 | 5.0 | 56.81 |
108 | 4.09 ± 0.21 | 2.74 × 1010 | 1.73 × 1012 | 8.58 ± 0.04 | 537 ± 75 | 23 | 13 | 4.3 | (–) |
120 | 3.88 ± 0.16 | 1.14 × 1010 | 1.96 × 1013 | 8.73 ± 0.02 | 712 ± 15 | 23 | 13 | 4.0 | (–) |
132 | 3.08 ± 0.19 | 2.16 × 109 | 3.71 × 1013 | 8.82 ± 0.03 | 502 ± 01 | (–) | (–) | (–) | (–) |
Fungal Phytopathogen | Reported Disease | Antifungal Activity | ||||
---|---|---|---|---|---|---|
Inhibition Diameter (mm) | MIC μg/mL | MFC μg/mL | Relation MFC/MIC | Action Mode | ||
Ascochyta sp. | Leaf blotch | 24 | 12.5 | 100 | 1 | Fungicide |
Colletotrichum acutatum | Anthracnose | 20 | 25 | 25 | 1 | Fungicide |
Colletotrichum gloeosporioides * | Anthracnose | 27 | 25 | 100 | 4 | Fungicide |
Colletotrichum gloeosporioides | Anthracnose | 28 | 25 | 100 | 4 | Fungicide |
Colletotrichum capsici | Anthracnose | 24 | 25 | 50 | 1 | Fungicide |
Curvularia clavata | Leaf spot | 25 | 400 | 400 | 4 | Fungicide |
Fusarium nivale | Fusariosis | 17 | 100 | 400 | 1 | Fungicide |
Fusarium solani | Fusariosis | 15.5 | 100 | 400 | 4 | Fungicide |
Moniliophthora roreri | Frosty pod rot | 34 | 100 | 400 | 4 | Fungicide |
Pestalotiopsis maculans | Leaf spot | 26 | 50 | 400 | 16 | Fungistatic |
Hydrocarbons | CL84 (%) | SDS | Triton 100-X | |
---|---|---|---|---|
Alkanes | n-Hexadecane | 57.79 | 58.33 | 56.48 |
n-Hexane | 54.90 | 58.33 | 57.84 | |
Crude oil (petroleum) | 50.98 | 47.56 | 47.56 | |
Olive Oil | 44.69 | 48.80 | 59.24 | |
Diesel (gas oil) | 59.00 | 65.21 | 57.50 | |
Burnt motor Oil | 66.67 | 49.41 | 75.93 | |
Motor Oil | 56.76 | 83.06 | 49.39 | |
Aromatics | Toluene | 61.90 | 60.71 | 62.20 |
Xylene | 59.95 | 61.18 | 61.63 |
pH | EI24 (%) | Salinity (NaCl %) | EI24 (%) |
---|---|---|---|
2 | 36.2 | 2 | 57.2 |
3 | 48.9 | 4 | 59.0 |
4 | 52.3 | 8 | 57.9 |
5 | 53.9 | 10 | 57.3 |
6 | 53.8 | 12 | 56.40 |
7 | 59.3 | Temperature (°C) | EI24 (%) |
8 | 65.0 | 0 | 37.2 |
9 | 60.0 | 5 | 55.3 |
10 | 51.6 | 70 | 56.2 |
11 | 48.3 | 100 | 60.8 |
12 | 56.6 | 120 | 60.4 |
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Guillén-Navarro, K.; López-Gutiérrez, T.; García-Fajardo, V.; Gómez-Cornelio, S.; Zarza, E.; De la Rosa-García, S.; Chan-Bacab, M. Broad-Spectrum Antifungal, Biosurfactants and Bioemulsifier Activity of Bacillus subtilis subsp. spizizenii—A Potential Biocontrol and Bioremediation Agent in Agriculture. Plants 2023, 12, 1374. https://doi.org/10.3390/plants12061374
Guillén-Navarro K, López-Gutiérrez T, García-Fajardo V, Gómez-Cornelio S, Zarza E, De la Rosa-García S, Chan-Bacab M. Broad-Spectrum Antifungal, Biosurfactants and Bioemulsifier Activity of Bacillus subtilis subsp. spizizenii—A Potential Biocontrol and Bioremediation Agent in Agriculture. Plants. 2023; 12(6):1374. https://doi.org/10.3390/plants12061374
Chicago/Turabian StyleGuillén-Navarro, Karina, Tomás López-Gutiérrez, Verónica García-Fajardo, Sergio Gómez-Cornelio, Eugenia Zarza, Susana De la Rosa-García, and Manuel Chan-Bacab. 2023. "Broad-Spectrum Antifungal, Biosurfactants and Bioemulsifier Activity of Bacillus subtilis subsp. spizizenii—A Potential Biocontrol and Bioremediation Agent in Agriculture" Plants 12, no. 6: 1374. https://doi.org/10.3390/plants12061374