Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (1): 253-263.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0449
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ZHANG Ling(), ZHANG Rong-yi, LIU Sheng-ke, TAN Zhi-qiong()
Received:
2022-04-12
Online:
2023-01-26
Published:
2023-02-02
Contact:
TAN Zhi-qiong
E-mail:2954424112@qq.com;990988@Hainanu.edu.cn
ZHANG Ling, ZHANG Rong-yi, LIU Sheng-ke, TAN Zhi-qiong. Screening of Antagonistic Bacteria for Bacterial Fruit Blotch of Cucurbits and Its Antibacterial Effects[J]. Biotechnology Bulletin, 2023, 39(1): 253-263.
类型Type | 引物Primer(5'-3') | 反应体系Reaction system | 循环条件Cyclic condition |
---|---|---|---|
16S rRNA | 27F: AGAGTTTGATCCTGGCTCAG 1492R: TACCTTGTTACGACTT | 2×Es Taq MasterMix 25 μL,ddH2O 19 μL,引物各2 μL,DNA模板2 μL | 95℃预变性5 min;94℃变性60 s,55℃退火90 s,72℃延伸1 min,共30个循环;72℃延伸10 min |
gyrB | UP-1S: GAAGTCATCATGACCGTTCTGC- AYGCNGGNGGNAARTTYGA UP-2Sr: AGCAGGGTACGGATGTGCGA- GCCRTCNACRTCNGCRTCNGTCAT | 2×Es Taq MasterMix 25 μL,ddH2O 21 μL,引物各1 μL,DNA模板2 μL | 94℃预变性10 min;94℃变性30 s,52℃退火30 s,72℃延伸1 min,共35个循环;72℃延伸10 min |
Table 1 PCR conditions
类型Type | 引物Primer(5'-3') | 反应体系Reaction system | 循环条件Cyclic condition |
---|---|---|---|
16S rRNA | 27F: AGAGTTTGATCCTGGCTCAG 1492R: TACCTTGTTACGACTT | 2×Es Taq MasterMix 25 μL,ddH2O 19 μL,引物各2 μL,DNA模板2 μL | 95℃预变性5 min;94℃变性60 s,55℃退火90 s,72℃延伸1 min,共30个循环;72℃延伸10 min |
gyrB | UP-1S: GAAGTCATCATGACCGTTCTGC- AYGCNGGNGGNAARTTYGA UP-2Sr: AGCAGGGTACGGATGTGCGA- GCCRTCNACRTCNGCRTCNGTCAT | 2×Es Taq MasterMix 25 μL,ddH2O 21 μL,引物各1 μL,DNA模板2 μL | 94℃预变性10 min;94℃变性30 s,52℃退火30 s,72℃延伸1 min,共35个循环;72℃延伸10 min |
病情等级 Disease grade | 分类标准 Classification criterion |
---|---|
0级Level 0 | 病斑数量为0 |
1级Level 1 | 总的叶片面积中病斑占比< 5% |
2级Level 2 | 5% ≤总的叶片面积中病斑占比< 15% |
3级Level 3 | 15% ≤总的叶片面积中病斑占比< 25% |
4级Level 4 | 25% ≤总的叶片面积中病斑占比< 35% |
5级Level 5 | 35% ≤总的叶片面积中病斑占比< 45% |
6级Level 6 | 45% ≤总的叶片面积中病斑占比< 55% |
7级Level 7 | 55% ≤总的叶片面积中病斑占比< 65% |
8级Level 8 | 65% ≤总的叶片面积中病斑占比< 75% |
9级Level 9 | 总的叶片面积中病斑占比≥75% |
Table 2 Classification criteria of disease grade
病情等级 Disease grade | 分类标准 Classification criterion |
---|---|
0级Level 0 | 病斑数量为0 |
1级Level 1 | 总的叶片面积中病斑占比< 5% |
2级Level 2 | 5% ≤总的叶片面积中病斑占比< 15% |
3级Level 3 | 15% ≤总的叶片面积中病斑占比< 25% |
4级Level 4 | 25% ≤总的叶片面积中病斑占比< 35% |
5级Level 5 | 35% ≤总的叶片面积中病斑占比< 45% |
6级Level 6 | 45% ≤总的叶片面积中病斑占比< 55% |
7级Level 7 | 55% ≤总的叶片面积中病斑占比< 65% |
8级Level 8 | 65% ≤总的叶片面积中病斑占比< 75% |
9级Level 9 | 总的叶片面积中病斑占比≥75% |
Fig. 2 Colony and Gram stain of strain 131 and 791 A and B are the colony map and Gram-stained map of strain 131,respectively;C and D are the colony map and Gram-stained map of strain 791,respectively
指标Index | 结果Result | |
---|---|---|
131 | 791 | |
革兰氏染色Gram stain | + | + |
接触酶试验Catalase test | + | + |
氧化酶试验Oxidase test | + | + |
厌氧生长Anaerobic growth | - | - |
2% NaCl | + | + |
10% NaCl | + | + |
明胶液化Gelatin liquefaction | + | + |
淀粉水解Amylohydrolysis | + | + |
甲基红试验MR | + | + |
V-P | + | + |
碳源利用Carbon source utilization | ||
棉子糖Raffinose | + | + |
半乳糖Galactose | + | + |
乳糖Lactose | + | + |
葡萄糖Glucose | + | + |
蔗糖Sucrose | + | + |
甘露醇Mannitol | + | + |
氮源利用Nitrogen source utilization | ||
硝酸铵Ammonium nitrate | + | + |
氯化铵Ammonium chloride | + | + |
尿素Urea | + | + |
硫酸铵Ammonium sulfate | + | + |
硝酸钾Potassium nitrate | + | + |
蛋白胨Peptone | + | + |
Table 3 Physiological and biochemical identification resu-lts of strain 131 and 791
指标Index | 结果Result | |
---|---|---|
131 | 791 | |
革兰氏染色Gram stain | + | + |
接触酶试验Catalase test | + | + |
氧化酶试验Oxidase test | + | + |
厌氧生长Anaerobic growth | - | - |
2% NaCl | + | + |
10% NaCl | + | + |
明胶液化Gelatin liquefaction | + | + |
淀粉水解Amylohydrolysis | + | + |
甲基红试验MR | + | + |
V-P | + | + |
碳源利用Carbon source utilization | ||
棉子糖Raffinose | + | + |
半乳糖Galactose | + | + |
乳糖Lactose | + | + |
葡萄糖Glucose | + | + |
蔗糖Sucrose | + | + |
甘露醇Mannitol | + | + |
氮源利用Nitrogen source utilization | ||
硝酸铵Ammonium nitrate | + | + |
氯化铵Ammonium chloride | + | + |
尿素Urea | + | + |
硫酸铵Ammonium sulfate | + | + |
硝酸钾Potassium nitrate | + | + |
蛋白胨Peptone | + | + |
Fig. 8 Effects of two biocontrol bacteria on the isolated leaves of bacterial fruit blotch of cantaloupe A:Leaf only inoculated with water. B:Leaf only inoculated with the BFB liquid. C and D:Leaves inoculated with the bacterial liquid of strain 131,791 and the BFB liquid,respectively. The results in the figure were counted on the seventh day after treatment
试验处理 Treatment | 病情指数 Disease index | 防治效果 Control efficacy/% |
---|---|---|
只喷水Spray water only | - | - |
只喷病菌菌液Spray BFB bacterial solution only | 77.41 ± 0.64a | - |
喷131菌液和病菌菌液 Spray 131 bacterial solution and BFB bacterial solution | 23.33 ± 1.11b | 69.86 |
喷791菌液和病菌菌液 Spray 791 bacterial solution and BFB bacterial solution | 17.04 ± 1.28c | 77.99 |
Table 4 Control efficacy of strain 131 and 791 in potting
试验处理 Treatment | 病情指数 Disease index | 防治效果 Control efficacy/% |
---|---|---|
只喷水Spray water only | - | - |
只喷病菌菌液Spray BFB bacterial solution only | 77.41 ± 0.64a | - |
喷131菌液和病菌菌液 Spray 131 bacterial solution and BFB bacterial solution | 23.33 ± 1.11b | 69.86 |
喷791菌液和病菌菌液 Spray 791 bacterial solution and BFB bacterial solution | 17.04 ± 1.28c | 77.99 |
Fig. 9 Control efficacy of two strains against BFB in potting A:Spray water only. B:Spray BFB bacterial solution only. C:Simultaneously spray 131 bacterial solution and BFB bacterial solution. D:Simultaneously spray 791 bacterial solution and BFB bacterial solution. The results in the figure were counted on the seventh day after treatment
病原真菌 Pathogenic fungus | 抑菌直径 Inhibition diameter/mm | 抑菌率 Inhibition rate/% | ||
---|---|---|---|---|
131 | 791 | 131 | 791 | |
小麦赤霉病病菌 F. graminearum | 0 | 10.50±0.50c | 0 | 12.35 |
火龙果炭疽病病菌 C. gloeosporioides | 7.10±0.10b | 11.00±0.41c | 8.35 | 12.94 |
黄瓜叶斑病病菌 P. cichorii | 8.40±0.63b | 21.25±0.75a | 9.88 | 25.00 |
甜瓜蔓枯病病菌 D. bryoniae | 0 | 16.25±0.63b | 0 | 19.12 |
荔枝霜疫霉 P. litchii | 12.00±1.15a | 14.00±1.63b | 14.12 | 16.47 |
Table 5 Inhibition percentage of strain 131,791 against other pathogen
病原真菌 Pathogenic fungus | 抑菌直径 Inhibition diameter/mm | 抑菌率 Inhibition rate/% | ||
---|---|---|---|---|
131 | 791 | 131 | 791 | |
小麦赤霉病病菌 F. graminearum | 0 | 10.50±0.50c | 0 | 12.35 |
火龙果炭疽病病菌 C. gloeosporioides | 7.10±0.10b | 11.00±0.41c | 8.35 | 12.94 |
黄瓜叶斑病病菌 P. cichorii | 8.40±0.63b | 21.25±0.75a | 9.88 | 25.00 |
甜瓜蔓枯病病菌 D. bryoniae | 0 | 16.25±0.63b | 0 | 19.12 |
荔枝霜疫霉 P. litchii | 12.00±1.15a | 14.00±1.63b | 14.12 | 16.47 |
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