Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (2): 277-288.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0918
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WANG Nan1,2(), LIAO Yong-qin1,2, SHI Zhu-feng2, SHEN Yun-xin1,2, YANG Tong-yu1,2, FENG Lu-yao2, YI Xiao-peng1,2, TANG Jia-cai1,2, CHEN Qi-bin1(), YANG Pei-wen2()
Received:
2023-09-25
Online:
2024-02-26
Published:
2024-03-13
Contact:
CHEN Qi-bin, YANG Pei-wen
E-mail:1164513193@qq.com;tclass99@163.com;398036877@qq.com
WANG Nan, LIAO Yong-qin, SHI Zhu-feng, SHEN Yun-xin, YANG Tong-yu, FENG Lu-yao, YI Xiao-peng, TANG Jia-cai, CHEN Qi-bin, YANG Pei-wen. Identification of Three Strains of Bacillus from the Forest Soil of Wuliang Mountain and Mining of Their Bioactivities[J]. Biotechnology Bulletin, 2024, 40(2): 277-288.
培养基种类 Medium category | 配方Formulation/L |
---|---|
马铃薯葡萄糖琼脂培养基 Potato dextrose agar medium(PDA) | 去皮马铃薯200.0 g,葡萄糖20.0 g,琼脂20.0 g,pH自然 |
营养琼脂培养基 Nutrient agar medium(NA) | 牛肉膏5.0 g,蛋白胨10.0 g,氯化钠5.0 g,琼脂20.0 g,pH 7.0 |
LB固体培养基 Luria-Bertani solid medium | 胰蛋白胨10.0 g,酵母提取物5.0 g,氯化钠10.0 g,琼脂20.0 g,pH 7.4 |
营养肉汤培养基 Nutrient broth medium(NB) | 牛肉膏3.0 g,蛋白胨10.0 g,氯化钠5.0 g,pH 7.4 |
水琼脂培养基 Water agar medium | 琼脂20.0 g,pH 7.0 |
解磷选择培养基 Phosphate-solubilizing selection medium | 葡萄糖10.0 g,硫酸铵0.5 g,氯化钠0.3 g,七水合硫酸镁0.3 g,七水合硫酸亚铁0.03 g,四水合硫酸锰0.03 g,氯化钾0.3 g,磷酸钙2.0 g,琼脂17.0 g,pH 7.2-7.4 |
解钾选择培养基 Potassium solution selection medium | 蔗糖5.0 g,葡萄糖5.0 g,硫酸铵0.5 g,酵母膏0.5 g,七水合硫酸镁0.3 g,磷酸氢二钠2.0 g,七水合硫酸亚铁0.03 g,硫酸锰0.03 g,钾长石粉2.0 g,琼脂18.0 g,pH 7.0 |
固氮选择培养基 Nitrogen-fixing selection medium | 磷酸二氢钾0.2 g,硫酸镁0.2 g,氯化钠0.2 g,碳酸钙5.0 g,甘露醇10.0 g,硫酸钙0.1 g,琼脂18.0 g,pH 6.8-7.0 |
酪蛋白选择培养基 | 葡萄糖10.0 g,酵母膏4.0 g,酪蛋白10.0 g,磷酸氢二钾1.0 g,七水合硫酸镁0.2 g,琼脂20.0 g,pH 7.0 |
纤维素培养基 Cellulose medium | 磷酸氢二钾1.0 g,硫酸铵2.0 g,七水合硫酸镁0.5 g,氯化钠0.5 g,微晶纤维素2.0 g,琼脂20.0 g,pH 7.0 |
淀粉选择培养基 Starch selection medium | 可溶性淀粉10.0 g,磷酸氢二钾1.0 g,硫酸镁1.0 g,氯化钠1.0 g,硫酸铵2.0 g,碳酸钙2.0 g,硫酸亚铁0.001 g,氯化锰0.001 g,硫酸锌0.001 g,琼脂20.0 g,pH 7.0-7.4 |
溶锌选择培养基[ Zinc-solubilized selection medium | 氧化锌1.0 g,葡萄糖10.0 g,氯化钾0.2 g、硫酸铵1.0 g,硫酸镁0.2 g,磷酸氢二钾0.1 g,琼脂20.0 g,pH 7.0 |
CAS双层显色培养基[ CAS bilayer chromogenic medium | 底层为5 mL水琼脂培养基(加有2 mL CAS显色液),上层为5 mL的PDA培养基 |
Table 1 Composition of test media
培养基种类 Medium category | 配方Formulation/L |
---|---|
马铃薯葡萄糖琼脂培养基 Potato dextrose agar medium(PDA) | 去皮马铃薯200.0 g,葡萄糖20.0 g,琼脂20.0 g,pH自然 |
营养琼脂培养基 Nutrient agar medium(NA) | 牛肉膏5.0 g,蛋白胨10.0 g,氯化钠5.0 g,琼脂20.0 g,pH 7.0 |
LB固体培养基 Luria-Bertani solid medium | 胰蛋白胨10.0 g,酵母提取物5.0 g,氯化钠10.0 g,琼脂20.0 g,pH 7.4 |
营养肉汤培养基 Nutrient broth medium(NB) | 牛肉膏3.0 g,蛋白胨10.0 g,氯化钠5.0 g,pH 7.4 |
水琼脂培养基 Water agar medium | 琼脂20.0 g,pH 7.0 |
解磷选择培养基 Phosphate-solubilizing selection medium | 葡萄糖10.0 g,硫酸铵0.5 g,氯化钠0.3 g,七水合硫酸镁0.3 g,七水合硫酸亚铁0.03 g,四水合硫酸锰0.03 g,氯化钾0.3 g,磷酸钙2.0 g,琼脂17.0 g,pH 7.2-7.4 |
解钾选择培养基 Potassium solution selection medium | 蔗糖5.0 g,葡萄糖5.0 g,硫酸铵0.5 g,酵母膏0.5 g,七水合硫酸镁0.3 g,磷酸氢二钠2.0 g,七水合硫酸亚铁0.03 g,硫酸锰0.03 g,钾长石粉2.0 g,琼脂18.0 g,pH 7.0 |
固氮选择培养基 Nitrogen-fixing selection medium | 磷酸二氢钾0.2 g,硫酸镁0.2 g,氯化钠0.2 g,碳酸钙5.0 g,甘露醇10.0 g,硫酸钙0.1 g,琼脂18.0 g,pH 6.8-7.0 |
酪蛋白选择培养基 | 葡萄糖10.0 g,酵母膏4.0 g,酪蛋白10.0 g,磷酸氢二钾1.0 g,七水合硫酸镁0.2 g,琼脂20.0 g,pH 7.0 |
纤维素培养基 Cellulose medium | 磷酸氢二钾1.0 g,硫酸铵2.0 g,七水合硫酸镁0.5 g,氯化钠0.5 g,微晶纤维素2.0 g,琼脂20.0 g,pH 7.0 |
淀粉选择培养基 Starch selection medium | 可溶性淀粉10.0 g,磷酸氢二钾1.0 g,硫酸镁1.0 g,氯化钠1.0 g,硫酸铵2.0 g,碳酸钙2.0 g,硫酸亚铁0.001 g,氯化锰0.001 g,硫酸锌0.001 g,琼脂20.0 g,pH 7.0-7.4 |
溶锌选择培养基[ Zinc-solubilized selection medium | 氧化锌1.0 g,葡萄糖10.0 g,氯化钾0.2 g、硫酸铵1.0 g,硫酸镁0.2 g,磷酸氢二钾0.1 g,琼脂20.0 g,pH 7.0 |
CAS双层显色培养基[ CAS bilayer chromogenic medium | 底层为5 mL水琼脂培养基(加有2 mL CAS显色液),上层为5 mL的PDA培养基 |
种类Category | 靶基因 Target gene | 引物序列 Primer sequence(5'-3') | 扩增长度 Amplification size/bp |
---|---|---|---|
Surfactins | Sfp | F:ATGAAGATTTACGGAATTTA R:TTATAAAAGCTCTTCGTACG | 675 |
srfA | F:ACACAGATATCAGGCAAGC R:GTCCCATCGTTCCTTCACA | 1 300 | |
Fengycins | fenA | F:GCTGTCCGTTCTGCTTTTTC R:GTCGGTGCATGAAATGTACG | 1 000 |
fenB | F:CTATAGTTTGTTGACGGCTC R:CAGCACTGGTTCTTGTCGCA | 1 600 | |
Iturins | ituA | F:ATGTATACCAGTCAATTCC R:GATCCGAAGCTGACAATAG | 1 047 |
ituC | F:TTCACTTTTGATCTGGCGAT R:CGTCCGGTACATTTTCAC | 575 | |
ituD | F:GATGCGATCTCCTTGGATGT R:ATCGTCATGTGCTGCTTGAG | 647 | |
Bacillomycin D | bymA | F:AAAGCGGCTCAAGAAGCGAAACCC R:CGATTCAGCTCATCGACCAGGTAGGC-3 | 1 200 |
bymB | F:AATAGAAGAACTGCTGGCGT R:GCCTTCCCGACACGACACT | 983 | |
bymC | F:GAAGGACACGGCAGAGAGGTC R:CACTGATGACTGTTCCTGCT | 875 |
Table 2 Detection primers for lipopeptide resistance genes
种类Category | 靶基因 Target gene | 引物序列 Primer sequence(5'-3') | 扩增长度 Amplification size/bp |
---|---|---|---|
Surfactins | Sfp | F:ATGAAGATTTACGGAATTTA R:TTATAAAAGCTCTTCGTACG | 675 |
srfA | F:ACACAGATATCAGGCAAGC R:GTCCCATCGTTCCTTCACA | 1 300 | |
Fengycins | fenA | F:GCTGTCCGTTCTGCTTTTTC R:GTCGGTGCATGAAATGTACG | 1 000 |
fenB | F:CTATAGTTTGTTGACGGCTC R:CAGCACTGGTTCTTGTCGCA | 1 600 | |
Iturins | ituA | F:ATGTATACCAGTCAATTCC R:GATCCGAAGCTGACAATAG | 1 047 |
ituC | F:TTCACTTTTGATCTGGCGAT R:CGTCCGGTACATTTTCAC | 575 | |
ituD | F:GATGCGATCTCCTTGGATGT R:ATCGTCATGTGCTGCTTGAG | 647 | |
Bacillomycin D | bymA | F:AAAGCGGCTCAAGAAGCGAAACCC R:CGATTCAGCTCATCGACCAGGTAGGC-3 | 1 200 |
bymB | F:AATAGAAGAACTGCTGGCGT R:GCCTTCCCGACACGACACT | 983 | |
bymC | F:GAAGGACACGGCAGAGAGGTC R:CACTGATGACTGTTCCTGCT | 875 |
菌株 Strain | 初筛抑菌率Inhibition rate of initial screening/% | 复筛抑菌率Inhibition rate of re-screening/% |
---|---|---|
SH-53 | 92.35±0.99a | 89.59±1.18a |
N4471 | 87.29±0.67b | 88.06±0.90a |
N9456 | 88.47±0.83b | 85.35±0.67a |
N1468 | 80.00±1.71cd | 75.41±1.23b |
N1475 | 82.35±1.03c | 80.59±0.68b |
N1477 | 74.47±1.52e | 64.71±1.33c |
82-a2 | 79.41±1.21d | 79.61±0.68b |
82-a3 | 77.06±1.33e | 74.70±1.56b |
SH-1 | 76.71±1.02e | 73.13±1.23b |
SH-6 | 75.29±1.55e | 58.04±2.45d |
Table 3 Results of screening functional strains
菌株 Strain | 初筛抑菌率Inhibition rate of initial screening/% | 复筛抑菌率Inhibition rate of re-screening/% |
---|---|---|
SH-53 | 92.35±0.99a | 89.59±1.18a |
N4471 | 87.29±0.67b | 88.06±0.90a |
N9456 | 88.47±0.83b | 85.35±0.67a |
N1468 | 80.00±1.71cd | 75.41±1.23b |
N1475 | 82.35±1.03c | 80.59±0.68b |
N1477 | 74.47±1.52e | 64.71±1.33c |
82-a2 | 79.41±1.21d | 79.61±0.68b |
82-a3 | 77.06±1.33e | 74.70±1.56b |
SH-1 | 76.71±1.02e | 73.13±1.23b |
SH-6 | 75.29±1.55e | 58.04±2.45d |
处理 Treatment | 株高 Height/cm | 茎粗 Stem thickness/mm | 根长 Root length/cm | 茎叶鲜重 Fresh weight of stems and leaves/g | 茎叶干重 Dry weight of stems and leaves/g | 根鲜重 Fresh weight of roots/g | 根干重 Dry weight of roots/g |
---|---|---|---|---|---|---|---|
Ck1 | 57.00±8.27b | 3.82±1.21b | 21.89±4.81b | 21.92±7.52b | 2.72±0.97b | 2.15±1.09b | 0.14±0.19a |
Ck2 | 62.50±9.06b | 5.24±1.38b | 25.41±8.24b | 22.44±8.96b | 3.94±2.09b | 2.87±1.14b | 0.21±0.21a |
SH-53 | 84.10±9.35a | 6.30±0.71a | 38.71±9.01a | 33.81±8.96a | 5.81±2.09a | 3.21±0.04b | 0.44±0.31a |
N4471 | 79.20±11.05a | 6.41±0.87a | 29.85±7.20b | 33.49±10.85a | 7.22±2.00a | 4.34±1.76a | 0.53±0.38a |
N9456 | 82.80±10.38a | 6.17±0.88a | 33.46±7.26a | 37.13±9.60a | 6.13±2.25a | 4.01±1.26a | 0.35±0.30a |
Table 4 Effects of functional strains on the growths of tomato seedlings
处理 Treatment | 株高 Height/cm | 茎粗 Stem thickness/mm | 根长 Root length/cm | 茎叶鲜重 Fresh weight of stems and leaves/g | 茎叶干重 Dry weight of stems and leaves/g | 根鲜重 Fresh weight of roots/g | 根干重 Dry weight of roots/g |
---|---|---|---|---|---|---|---|
Ck1 | 57.00±8.27b | 3.82±1.21b | 21.89±4.81b | 21.92±7.52b | 2.72±0.97b | 2.15±1.09b | 0.14±0.19a |
Ck2 | 62.50±9.06b | 5.24±1.38b | 25.41±8.24b | 22.44±8.96b | 3.94±2.09b | 2.87±1.14b | 0.21±0.21a |
SH-53 | 84.10±9.35a | 6.30±0.71a | 38.71±9.01a | 33.81±8.96a | 5.81±2.09a | 3.21±0.04b | 0.44±0.31a |
N4471 | 79.20±11.05a | 6.41±0.87a | 29.85±7.20b | 33.49±10.85a | 7.22±2.00a | 4.34±1.76a | 0.53±0.38a |
N9456 | 82.80±10.38a | 6.17±0.88a | 33.46±7.26a | 37.13±9.60a | 6.13±2.25a | 4.01±1.26a | 0.35±0.30a |
测定项目 Measuring item | 反应 Reaction | |||
---|---|---|---|---|
SH-53 | N4471 | N9456 | ||
革兰氏染色 Gram staining | + | + | + | |
形成芽孢Forming spores | + | + | + | |
菌体形态 Mycelial morphology | Rhabditiform | Rhabditiform | Rhabditiform | |
接触酶 Catalase | + | + | + | |
V-P 实验V-P experiment | + | + | + | |
甲基红 Methyl red | - | - | - | |
分解淀粉 Gelatin liquefaction | + | + | + | |
最低生长温度 Minimum growth temperature | 4℃ | 4℃ | 4℃ | |
生长含盐量范围 Growth salinity range | 0%-13% | 0%-13% | 0%-14% | |
产吲哚Producing indoles | + | + | - | |
厌氧生长 Anaerobic growth | - | - | - | |
硝化酸盐还原 Nitrate reduction | + | + | + |
Table 5 Determination of physiological and biochemical functions of the functional strains
测定项目 Measuring item | 反应 Reaction | |||
---|---|---|---|---|
SH-53 | N4471 | N9456 | ||
革兰氏染色 Gram staining | + | + | + | |
形成芽孢Forming spores | + | + | + | |
菌体形态 Mycelial morphology | Rhabditiform | Rhabditiform | Rhabditiform | |
接触酶 Catalase | + | + | + | |
V-P 实验V-P experiment | + | + | + | |
甲基红 Methyl red | - | - | - | |
分解淀粉 Gelatin liquefaction | + | + | + | |
最低生长温度 Minimum growth temperature | 4℃ | 4℃ | 4℃ | |
生长含盐量范围 Growth salinity range | 0%-13% | 0%-13% | 0%-14% | |
产吲哚Producing indoles | + | + | - | |
厌氧生长 Anaerobic growth | - | - | - | |
硝化酸盐还原 Nitrate reduction | + | + | + |
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