Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (4): 228-241.doi: 10.13560/j.cnki.biotech.bull.1985.2023-1172
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XU Wei-fang1,2(), LI He-yu1, ZHANG Hui1, HE Zi-ang1, GAO Wen-heng1, XIE Zi-yang1, WANG Chuan-wen3, YIN Deng-ke1()
Received:
2023-12-13
Online:
2024-04-26
Published:
2024-04-30
Contact:
YIN Deng-ke
E-mail:xuweifang@ahtcm.edu.cn;yindengke@ahtcm.edu.cn
XU Wei-fang, LI He-yu, ZHANG Hui, HE Zi-ang, GAO Wen-heng, XIE Zi-yang, WANG Chuan-wen, YIN Deng-ke. Efficacy and Its Mechanism of Bacterial Strain HX0037 on the Control of Anthracnose Disease of Trichosanthes kirilowii Maxim[J]. Biotechnology Bulletin, 2024, 40(4): 228-241.
培养基名称Name of culture medium | 组分Component/(g·L-1) |
---|---|
马铃薯葡萄糖培养基Potato dextrose medium, PDB | 马铃薯200.0,葡萄糖20.0 |
LB培养基Luria-Bertani medium | 胰蛋白胨10.0,酵母粉5.0,氯化钠10.0 |
PDB改良培养基Modified potato dextrose broth | 马铃薯200.0,麦芽糖20.0,蛋白胨10.0,硫酸铵5.0,磷酸氢二钠1.5 |
纤维素酶检测培养基Cellulase assay medium | 羧甲基纤维素钠 2.0,硫酸铵 2.0,磷酸二氢钾 1.0,硫酸镁 0.5,刚果红 0.1 |
蛋白酶检测培养基Protease assay medium | 葡萄糖15.0,脱脂奶粉20.0,氯化钾 0.5,硫酸镁 0.5 |
淀粉酶检测培养基Amylase assay medium | 牛肉膏5.0,蛋白胨10.0,氯化钠 5.0,可溶性淀粉10.0 |
铁载体检测培养基主要成分Siderophore assay medium | 葡萄糖4.0,硝酸钾0.2,硫酸镁 0.1,氯化钠0.1,磷酸氢二钾 0.1,硫酸亚铁 0.002 |
Landy发酵培养基Landy fermentation medium | 葡萄糖20.0,谷氨酸钠 5.0, 酵母粉1.0, 磷酸二氢钾 1.0,硫酸镁 0.5,氯化钾 0.5,硫酸锰 0.000 05,硫酸铜 0.000 16,硫酸亚铁 0.000 15,苯丙氨酸 0.02 |
Table 1 Formulations of media required for the experiments
培养基名称Name of culture medium | 组分Component/(g·L-1) |
---|---|
马铃薯葡萄糖培养基Potato dextrose medium, PDB | 马铃薯200.0,葡萄糖20.0 |
LB培养基Luria-Bertani medium | 胰蛋白胨10.0,酵母粉5.0,氯化钠10.0 |
PDB改良培养基Modified potato dextrose broth | 马铃薯200.0,麦芽糖20.0,蛋白胨10.0,硫酸铵5.0,磷酸氢二钠1.5 |
纤维素酶检测培养基Cellulase assay medium | 羧甲基纤维素钠 2.0,硫酸铵 2.0,磷酸二氢钾 1.0,硫酸镁 0.5,刚果红 0.1 |
蛋白酶检测培养基Protease assay medium | 葡萄糖15.0,脱脂奶粉20.0,氯化钾 0.5,硫酸镁 0.5 |
淀粉酶检测培养基Amylase assay medium | 牛肉膏5.0,蛋白胨10.0,氯化钠 5.0,可溶性淀粉10.0 |
铁载体检测培养基主要成分Siderophore assay medium | 葡萄糖4.0,硝酸钾0.2,硫酸镁 0.1,氯化钠0.1,磷酸氢二钾 0.1,硫酸亚铁 0.002 |
Landy发酵培养基Landy fermentation medium | 葡萄糖20.0,谷氨酸钠 5.0, 酵母粉1.0, 磷酸二氢钾 1.0,硫酸镁 0.5,氯化钾 0.5,硫酸锰 0.000 05,硫酸铜 0.000 16,硫酸亚铁 0.000 15,苯丙氨酸 0.02 |
病原菌 Pathogenic bacterium | 抑菌率 Inhibition rate/%(x±s, n=3) |
---|---|
栝楼炭疽病菌C. gloeosporioides | 57.59±0.00a |
梨炭疽病菌C. fructicola | 38.97±0.01c |
枣炭疽病菌C. coccodes | 48.54±0.01b |
苹果炭疽病菌C. malicorticis | 34.27±0.01d |
Table 2 Antagonistic activity of strain HX0037 against four pathogens of fruit anthracnose disease
病原菌 Pathogenic bacterium | 抑菌率 Inhibition rate/%(x±s, n=3) |
---|---|
栝楼炭疽病菌C. gloeosporioides | 57.59±0.00a |
梨炭疽病菌C. fructicola | 38.97±0.01c |
枣炭疽病菌C. coccodes | 48.54±0.01b |
苹果炭疽病菌C. malicorticis | 34.27±0.01d |
Fig. 1 Antagonistic activity of strain HX0037 against C. gloeosporioides(Bars=10 µm) A: Colony morphology of C. gloeosporioides on PDA medium(A1: Normal C. gloeosporioides; A2: C. gloeosporioides co-cultured with HX0037 cell suspension; A3: C. gloeosporioides cultured on PDA medium containing HX0037 cell-free fermentation broth). B: PI staining of C. gloeosporioides treated with HX0037 cell suspension
检测项目 Test item | 检测结果 Test result |
---|---|
葡萄糖(产酸产气)Glucose(Producing acid and gas) | + |
乳糖 Lactose | + |
麦芽糖 Maltose | - |
甘露醇 Mannitol | - |
蔗糖 Sucrose | + |
蛋白胨水 Peptone water | - |
V-P试验Voges-Proskauer reaction | + |
西蒙氏柠檬酸盐 Symon's phosphate | + |
硫化氢 H2S production | - |
尿素 Urea | - |
半固体琼脂Semi-solid agar | - |
Table 3 Physiological and biochemical detection of strain HX0037
检测项目 Test item | 检测结果 Test result |
---|---|
葡萄糖(产酸产气)Glucose(Producing acid and gas) | + |
乳糖 Lactose | + |
麦芽糖 Maltose | - |
甘露醇 Mannitol | - |
蔗糖 Sucrose | + |
蛋白胨水 Peptone water | - |
V-P试验Voges-Proskauer reaction | + |
西蒙氏柠檬酸盐 Symon's phosphate | + |
硫化氢 H2S production | - |
尿素 Urea | - |
半固体琼脂Semi-solid agar | - |
基因类型 Type | 数目 Number | 长度 Length/bp | 占基因组大小 Proportion in genome/% |
---|---|---|---|
CDS | 3 712 | 3 436 344 | 88.38 |
tRNA | 82 | 6 330 | 0.16 |
16S rRNA | 9 | 13 929 | 0.36 |
23S rRNA | 9 | 26 370 | 0.68 |
5S rRNA | 9 | 1 044 | 0.03 |
Table 4 General situation of genome structure of strain HX0037
基因类型 Type | 数目 Number | 长度 Length/bp | 占基因组大小 Proportion in genome/% |
---|---|---|---|
CDS | 3 712 | 3 436 344 | 88.38 |
tRNA | 82 | 6 330 | 0.16 |
16S rRNA | 9 | 13 929 | 0.36 |
23S rRNA | 9 | 26 370 | 0.68 |
5S rRNA | 9 | 1 044 | 0.03 |
基因簇Cluster | 类型Type | 开始From | 结束To | 最相似的基因簇 The most similar gene cluster | 相似度Similarity/% |
---|---|---|---|---|---|
Cluster1 | NRPS, betalactone | 1 | 101 972 | Most similar known cluster | 86 |
Cluster2 | TransAT-PKS | 165 428 | 266 126 | Fengycin | 100 |
Cluster3 | TransAT-PKS | 490 930 | 577 278 | Bacillaene | 100 |
Cluster4 | Terpene | 897 298 | 913 914 | Macrolactin | - |
Cluster5 | PKS-like | 998 755 | 1 039 999 | - | 7 |
Cluster6 | NRPS | 1 570 321 | 1 635 119 | ButirosinAB | 82 |
Cluster7 | Lanthipeptide-class-iii | 1 730 700 | 1 753 315 | Surfactin | 35 |
Cluster8 | Other | 2 237 949 | 2 279 367 | Locillomycin | 100 |
Cluster9 | Pipp-like,NRPS | 2 809 532 | 2 860 040 | Bacilysin | 100 |
Cluster10 | TransAT-PKS | 3 480 099 | 3 573 848 | Bacillibactin | 100 |
Cluster11 | T3PKS | 3 701 691 | 3 742 791 | Difficidin | - |
Cluster12 | Terpene | 3 806 279 | 3 828 162 | - | - |
Table 5 Secondary metabolite synthesis gene cluster of strain HX0037
基因簇Cluster | 类型Type | 开始From | 结束To | 最相似的基因簇 The most similar gene cluster | 相似度Similarity/% |
---|---|---|---|---|---|
Cluster1 | NRPS, betalactone | 1 | 101 972 | Most similar known cluster | 86 |
Cluster2 | TransAT-PKS | 165 428 | 266 126 | Fengycin | 100 |
Cluster3 | TransAT-PKS | 490 930 | 577 278 | Bacillaene | 100 |
Cluster4 | Terpene | 897 298 | 913 914 | Macrolactin | - |
Cluster5 | PKS-like | 998 755 | 1 039 999 | - | 7 |
Cluster6 | NRPS | 1 570 321 | 1 635 119 | ButirosinAB | 82 |
Cluster7 | Lanthipeptide-class-iii | 1 730 700 | 1 753 315 | Surfactin | 35 |
Cluster8 | Other | 2 237 949 | 2 279 367 | Locillomycin | 100 |
Cluster9 | Pipp-like,NRPS | 2 809 532 | 2 860 040 | Bacilysin | 100 |
Cluster10 | TransAT-PKS | 3 480 099 | 3 573 848 | Bacillibactin | 100 |
Cluster11 | T3PKS | 3 701 691 | 3 742 791 | Difficidin | - |
Cluster12 | Terpene | 3 806 279 | 3 828 162 | - | - |
Fig. 5 Phylogenetic tree of HX0037 strain based on 16S rDNA(A)and gyrA(B)sequence Numbers in parentheses is the GenBank accession numbers. Numbers at the nodes mean the Bootstrap value. The scale bar indicates 0.5% and 10% nucleotide substitution
菌株Strain | GenBank登录号GenBank No. | 平均核苷酸一致性ANI/% | GC含量GC conten t /% | 大小Size/bp |
---|---|---|---|---|
B. amyloliquefaciens L-H15 | CP010556 | 98.56 | 46.60 | 3 905 973 |
B. amyloliquefaciens S499 | CP014700 | 98.55 | 46.60 | 3 927 922 |
B. amyloliquefaciens EA19 | CP079834 | 98.55 | 40.20 | 3 964 177 |
B. amyloliquefaciens FZB42 | CP000560 | 97.78 | 46.48 | 3 918 596 |
B. subtilis BSD-2 | CP013654 | 79.91 | 43.88 | 4 030 837 |
B. subtilis 7PJ-16 | CP023409 | 79.87 | 43.28 | 4 209 045 |
B. licheniformis T5 | CP124852 | 76.48 | 46.16 | 4 247 430 |
B. licheniformis TAB7 | CP027789 | 76.46 | 45.82 | 4 367 367 |
B. megaterium HGS7 | CP065213 | 74.48 | 38.27 | 5 035 031 |
B. megaterium QM B1551 | CP001983 | 74.44 | 38.20 | 5 097 129 |
B. cereus ATCC 14579 | NC004722 | 74.23 | 35.30 | 5 097 129 |
Table 6 Comparisons ANI analysis of strain HX0037 and related Bacillus spp.
菌株Strain | GenBank登录号GenBank No. | 平均核苷酸一致性ANI/% | GC含量GC conten t /% | 大小Size/bp |
---|---|---|---|---|
B. amyloliquefaciens L-H15 | CP010556 | 98.56 | 46.60 | 3 905 973 |
B. amyloliquefaciens S499 | CP014700 | 98.55 | 46.60 | 3 927 922 |
B. amyloliquefaciens EA19 | CP079834 | 98.55 | 40.20 | 3 964 177 |
B. amyloliquefaciens FZB42 | CP000560 | 97.78 | 46.48 | 3 918 596 |
B. subtilis BSD-2 | CP013654 | 79.91 | 43.88 | 4 030 837 |
B. subtilis 7PJ-16 | CP023409 | 79.87 | 43.28 | 4 209 045 |
B. licheniformis T5 | CP124852 | 76.48 | 46.16 | 4 247 430 |
B. licheniformis TAB7 | CP027789 | 76.46 | 45.82 | 4 367 367 |
B. megaterium HGS7 | CP065213 | 74.48 | 38.27 | 5 035 031 |
B. megaterium QM B1551 | CP001983 | 74.44 | 38.20 | 5 097 129 |
B. cereus ATCC 14579 | NC004722 | 74.23 | 35.30 | 5 097 129 |
质量峰 Mass peak(m/z) | 碎片离子 Assignment | 参考文献 Reference |
---|---|---|
1 030.64 | Surfactin A C13[M+Na]+ | [ |
Surfactin B C14[M+Na]+ | [ | |
1 044.64 | Surfactin A C14[M+Na]+ | [ |
Surfactin B C15[M+Na]+ | [ | |
1 058.67 | Surfactin A C15[M+Na]+ | [ |
1 072.69 | Surfactin A C16[M+Na]+ | [ |
1 008.66 | Surfactin A C13[M+H]+ | [ |
1 022.67 | Surfactin A C14[M+H]+ | [ |
1 036.69 | Surfactin A C15[M+H]+ | [ |
1 050.71 | Surfactin A C16[M+H]+ | [ |
1 463.81 | Fengycin B C14[M+H]+ | [ |
Fengycin A C14[M+H]+ | [ | |
1 477.82 | Fengycin B C15[M+H]+ | [ |
Fengycin A C15[M+H]+ | [ | |
1 491.84 | Fengycin B C16[M+H]+ | [ |
Fengycin A C16[M+H]+ | [ |
Table 7 HPLC-MS analysis of lipopeptide produced by strain HX0037
质量峰 Mass peak(m/z) | 碎片离子 Assignment | 参考文献 Reference |
---|---|---|
1 030.64 | Surfactin A C13[M+Na]+ | [ |
Surfactin B C14[M+Na]+ | [ | |
1 044.64 | Surfactin A C14[M+Na]+ | [ |
Surfactin B C15[M+Na]+ | [ | |
1 058.67 | Surfactin A C15[M+Na]+ | [ |
1 072.69 | Surfactin A C16[M+Na]+ | [ |
1 008.66 | Surfactin A C13[M+H]+ | [ |
1 022.67 | Surfactin A C14[M+H]+ | [ |
1 036.69 | Surfactin A C15[M+H]+ | [ |
1 050.71 | Surfactin A C16[M+H]+ | [ |
1 463.81 | Fengycin B C14[M+H]+ | [ |
Fengycin A C14[M+H]+ | [ | |
1 477.82 | Fengycin B C15[M+H]+ | [ |
Fengycin A C15[M+H]+ | [ | |
1 491.84 | Fengycin B C16[M+H]+ | [ |
Fengycin A C16[M+H]+ | [ |
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