New Strain of Bacillus amyloliquefaciens G1 as a Potential Downy Mildew Biocontrol Agent for Grape
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.1.1. Isolation of Bacteria from Stems
2.1.2. Separation of Bacteria from Fruits
2.1.3. Separation of Bacteria from Seeds
2.2. Inhibition of Disease by Endogenous Bacteria
2.2.1. Preparation of Biocontrol Bacterial Suspension
2.2.2. Preparation of Grape Downy Mildew Sporangia
2.3. Determination of Endophytic Antagonistic Bacteria in the Field
2.4. Identification of Endogenous Antagonistic Bacteria
2.4.1. Morphological Analysis
2.4.2. S rDNA Sequence Analysis
2.5. Detection of Activity of Endogenous Antagonistic Enzymes and Secondary Metabolites
2.5.1. Detection of Protease Activity
2.5.2. Cellulase Activity Test
2.5.3. Detection of Siderophore Activity
2.6. Extraction of Antifungal Crude Protein from Antagonistic Strain G1
2.7. Determination of Antibacterial Activity of Protein
Determination of Bacterial Inhibitory Activity Using the Agar Well Diffusion Standoff Method
2.8. Purification and Identification of Strain G1 Antifungal Protein
2.8.1. Q-Sepharose FF Anion Exchange Chromatography
2.8.2. Identification of Proteins by LC-MS
2.9. Data Retrieval
3. Results
3.1. Isolation of Endophytic Bacteria from Grape Tissue
3.2. Endogenous Strains Capable of Inhibiting Downy Mildew
3.3. Effects of Endophytic Antagonistic Strains on Downy Mildew in the Field
3.4. Identification of G1
3.4.1. Morphological Examination
3.4.2. 16S rDNA Sequence Analysis
3.5. Enzyme Activity and Secondary Metabolites of G1
3.6. Antibacterial Activity of Crude Protein from G1
3.6.1. Extraction of Crude Protein
3.6.2. Inhibitory Effects of G1 against Pathogenic Fungi
3.6.3. Effects of G1 on Spore Germination
3.7. Purification and Identification of Antifungal Protein
3.7.1. Q-Sepharose FF Anion Exchange Chromatography
3.7.2. Identification of Proteins by LC-MS
4. Discussion and Conclusions
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Grape Varieties | Plant Tissue | Number of Strains/Strains | Strain Number |
---|---|---|---|
Vitis labrusca × vinifera ‘Kyoho | Seed | 5 | G30–G32, G34–G35 |
Manicure Finger grapes | Old stems | 6 | G24–G29 |
Vitis vinifera × V. labrusca ‘Boxin 1’ | Old stems | 12 | G13–G23, G36 |
Vitis vinifera ‘Hongru’ | Old stems | 1 | G37 |
Red Globe | Fruit | 12 | G1–G10, G33, G38 |
Red Globe | Seed | 1 | G11 |
Centennial Seedless | Fruit | 1 | G12 |
Numbering | Incidence Rate/% | Inhibition Rate/% | Strain Number | Incidence Rate/% | Inhibition Rate/% |
---|---|---|---|---|---|
CK | 87.5 ± 2.9 lmnop | — | G19 | 60.8 ± 9.6 efghi | 30.5 ± 11.0 efgh |
CN181 | 0.0 ± 0.0 a | 100.0 ± 0.0 a | G20 | 65.0 ± 10.1 fghij | 25.7 ± 11.5 fghij |
G1 | 0.00 ± 0.00 a | 100.0 ± 0.0 a | G21 | 55.8 ± 15.0 defgh | 36.2 ± 17.2 defg |
G2 | 46.7 ± 5.1 cdefg | 46.7 ± 5.8 cdef | G22 | 77.5 ± 5.8 io | 11.4 ± 6.6 hijklm |
G3 | 4.2 ± 2.2 a | 95.2 ± 2.5 a | G23 | 44.2 ± 8.7 cde | 49.5 ± 9.9 cde |
G4 | 1.7 ± 0.8 a | 98.1 ± 1.0 a | G24 | 61.7 ± 16.7 efghi | 29.5 ± 19.1 efghi |
G5 | 0.0 ± 0.0 a | 100.0 ± 0.0 a | G25 | 59.2 ± 3.0 efghi | 32.4 ± 3.4 efgh |
G6 | 7.5 ± 5.2 a | 91.4 ± 5.9 a | G26 | 45.8 ± 6.0 cdef | 47.6 ± 6.7 cdef |
G7 | 5.8 ± 3.0 a | 93.3 ± 3.4 a | G27 | 76.7 ± 5.2 in | 12.4 ± 5.8 hijklm |
G8 | 3.3 ± 2.2 a | 96.2 ± 2.5 a | G28 | 85.0 ± 3.8 klmnop | 2.9 ± 4.4 jklmn |
G9 | 0.0 ± 0.0 a | 100.0 ± 0.0 a | G29 | 65.8 ± 15.2 ghik | 24.8 ± 17.3 fghij |
G10 | 81.7 ± 0.8 jklmnop | 6.7 ± 1.0 ijklmn | G30 | 96.7 ± 0.8 op | −10.5 ± 1.0 mn |
G11 | 68.3 ± 11.0 hil | 21.9 ± 12.6 ghijk | G31 | 86.7 ± 4.4 lmnop | 1.0 ± 5.0 klmn |
G12 | 88.3 ± 6.7 mnop | −1.0 ± 7.6 klmn | G32 | 77.5 ± 2.9 io | 11.4 ± 3.3 hijklm |
G13 | 94.2 ± 3.0 mnop | −7.6 ± 3.4 lmn | G33 | 1.7 ± 1.7 a | 98.1 ± 1.9 a |
G14 | 75.8 ± 6.5 im | 13.3 ± 7.4 ghijkl | G34 | 100.0 ± 0.0 p | −14.3 ± 0.0 n |
G15 | 37.5 ± 6.6 cd | 57.1 ± 7.6 bcd | G35 | 96.7 ± 3.3 op | −10.5 ± 3.4 mn |
G16 | 34.2 ± 4.4 bc | 61.0 ± 5.0 bc | G36 | 98.3 ± 1.7 p | −12.4 ± 1.9 n |
G17 | 60.0 ± 12.6 efghi | 31.4 ± 14.4 efgh | G37 | 95.8 ± 4.2 nop | −9.5 ± 4.8 lmn |
G18 | 59.2 ± 11.6 efghi | 32.4 ± 13.2 efgh | G38 | 17.5 ± 10.9 ab | 80.0 ± 12.5 ab |
Time/h | Germination Rate of Control/% | Germination Rate of Treatment/% | Inhibitory Rate/% |
---|---|---|---|
2 | 6.40 ± 1.27 f | 3.15 ± 0.50 c | 50.78 |
4 | 28.01 ± 2.98 e | 4.71 ± 0.56 c | 83.18 |
6 | 52.61 ± 1.27 d | 5.65 ± 0.95 c | 89.26 |
8 | 71.97 ± 1.47 c | 20.34 ± 0.96 b | 71.74 |
10 | 86.01 ± 0.98 b | 33.67 ± 1.57 a | 60.85 |
12 | 91.96 ± 1.21 a | 32.35 ± 1.51 a | 64.82 |
Sporangium | Point Sample | Morbidity | INCIDENCE/% | Inhibition Rate/% |
---|---|---|---|---|
Mixture of crude protein and sporangium suspension (1:1) | 120 | 20 | 16.7% | 70.8 |
Sporangium of downy mildew | 120 | 105 | 87.5 |
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Qiao, W.; Kang, X.; Ma, X.; Ran, L.; Zhen, Z. New Strain of Bacillus amyloliquefaciens G1 as a Potential Downy Mildew Biocontrol Agent for Grape. Agronomy 2024, 14, 1532. https://doi.org/10.3390/agronomy14071532
Qiao W, Kang X, Ma X, Ran L, Zhen Z. New Strain of Bacillus amyloliquefaciens G1 as a Potential Downy Mildew Biocontrol Agent for Grape. Agronomy. 2024; 14(7):1532. https://doi.org/10.3390/agronomy14071532
Chicago/Turabian StyleQiao, Wenyan, Xingjiao Kang, Xiwei Ma, Longxian Ran, and Zhixian Zhen. 2024. "New Strain of Bacillus amyloliquefaciens G1 as a Potential Downy Mildew Biocontrol Agent for Grape" Agronomy 14, no. 7: 1532. https://doi.org/10.3390/agronomy14071532