Identification of an antagonistic strain Bacillus amyloliquefaciens E3 against Dickeya zeae and its antimicrobial activity
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摘要:目的
纯化和鉴定从柑橘根际土壤分离得到的可抑制水稻基腐病菌Dickeya zeae EC1生长的解淀粉芽孢杆菌Bacillus amyloliquefaciens，并解析其抑菌机制。
方法采用平板扩散法从根际土壤中筛选出1株拮抗D. zeae EC1的B. amyloliquefaciens E3菌株；根据细菌菌落表型、生理生化特征结合16S rDNA序列分析等方法对E3菌株进行分类鉴定；检测无菌E3培养液对D. zeae EC1侵染水稻种子能力的影响；盐酸沉淀结合丙酮抽提法提取E3菌株脂肽粗提物；采用平板对峙法测定E3菌株脂肽粗提物对病原真菌的抑菌活性，琼脂扩散法测定E3菌株脂肽粗提物对病原细菌的抑菌活性及最小抑菌浓度(Minimum inhibitory concentration, MIC)；用Durashell C18柱对脂肽粗提物进行HPLC分离纯化；通过液相色谱−质谱联用(LC-ESI-MS)分析，鉴定抑菌物质的相对分子质量并推测其化学组成。
结果B. amyloliquefaciens E3菌株对茄病镰刀菌Fusarium solani、香蕉基腐病菌、茄科劳尔氏菌Ralstonia solanacearum等多种植物病原菌生长具有抑制作用，表现为广谱抗性；B. amyloliquefaciens E3菌株的无菌培养液能够抑制D. zeae EC1侵染萌发的水稻种子，显著提高水稻种子的萌芽率；B. amyloliquefaciens E3菌株的脂肽粗提物可以抑制D. zeae EC1的生长，其MIC为348.97 µg/mL；HPLC分离纯化结合LC-ESI-MS分析发现，B. amyloliquefaciens E3菌株分泌的主要抑菌活性物质包括Surfactin、Fengycin和Iturin 3类脂肽类抗生素。
结论B. amyloliquefaciens E3菌株具有作为生防菌的潜力，它可能通过分泌Surfactin、Fengycin和Iturin 3种脂肽类抑菌活性物质，拮抗水稻基腐病菌、茄科劳尔氏菌、茄病镰刀菌等多种植物病原菌。研究结果可为该菌的生物防治应用提供理论依据。
Abstract:ObjectiveTo isolate and identify Bacillus amyloliquefaciens strain which can inhibit the growth of Dickeya zeae EC1 from the rhizosphere soil of citrus plants, and analyze its antibacterial mechanism.
MethodThe B. amyloliquefaciens E3 strain antagonizing the growth of D. zeae EC1 was screened from the rhizosphere soil by plate diffusion method. The E3 strain was classified and identified based on colony phenotype, physiological and biochemical characteristics of bacteria and 16S rDNA sequence analysis. Effects of E3 sterile culture medium on the seeds germination ability of rice infected by D. zeae EC1 was detected. The crude lipopeptides of E3 strain was extracted by hydrochloric acid precipitation combined with acetone extraction. The antimicrobial activity of crude lipopeptides against pathogenic fungi were determined by plate confrontation method. The antimicrobial activity of crude lipopetides against pathogenic bacteria were determined by agar diffusion method, and the minimum inhibitory concentration (MIC) was obtained. The crude lipopeptides were separated and purified by HPLC with Durashell C18 column. Liquid chromatography-mass spectrometry (LC-ESI-MS) analysis was used to identify the molecular weights of the antimicrobial substances and speculate its chemical composition.
ResultB. amyloliquefaciens E3 strain had an inhibitory effect against Fusarium solani, D. zeae MS1 and Ralstonia solanacearum, etc, indicating that it had broad-spectrum resistance. The sterile culture medium of B. amyloliquefaciens E3 strain inhibited the infection of germinating rice seeds by D. zeae EC1 and significantly increased the seed germination rate. The crude lipopeptides of E3 strain inhibited the growth of D. zeae EC1 with the MIC of 348.97 µg/mL. HPLC combined with LC-ESI-MS analysis showed that the main antibacterial substances secreted by E3 strain included surfactin, fengycin and iturin.
ConclusionThe B. amyloliquefaciens E3 strain is potential to be a biocontrol agent. It can antigonize a variety of plant pathogens such as D. zeae EC1, R. solanacearum and F. solani by secreting three kinds of lipopeptides including surfactin, fengycin and iturin. The results provide a theoretical basis for the application of B. amyloliquefaciens E3 in biological control.
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图 2 依据16S rDNA序列构建的E3菌株系统发育树
Figure 2. The phylogenetic tree of E3 strain based on 16S rDNA sequence
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图 3 β-甘露聚糖酶基因序列特异性扩增电泳结果
M：DS 5000 marker；1：16S rDNA；2：枯草芽孢杆菌β-甘露聚糖酶基因；3：解淀粉芽孢杆菌β-甘露聚糖酶基因
Figure 3. The electrophoresis results of specific amplification of β-mannanase gene sequence
M：DS 5000 marker；1：16S rDNA；2：β-mannanase gene from Bacillus subtilis；3：β-mannanase gene from B. amyloliquefaciens
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图 4 以无细胞上清液的抗菌活性为指标的培养基优化结果
Figure 4. Results of medium optimization based on the antibacterial activity of cell-free supernatant
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图 5 Bacillus amyloliquefacions E3菌株无菌培养液对Dickeya zeae EC1抑制水稻种子萌芽率的拮抗效果
“*”、“**”、“***”分别表示0.05、0.01和0.000 1水平的差异显著性(t检验)
Figure 5. Antagonism effect of the sterile culture medium of Bacillus amyloliquefaciens E3 strain on the inhibition of rice seeds germination by Dickeya zeae EC1
“*”，“**”，“***” indicate significance differences at the levels of 0.05，0.01 and 0.000 1 respectively (t test)
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图 6 Bacillus amyloliquefaciens E3菌株粗提脂肽的抑菌活性
A：甲醇；B：无菌上清液；C：粗提脂肽类物质
Figure 6. Antimicrobial activity of crude lipopeptides extracted from Bacillus amyloliquefaciens E3 strain
A：Methanol；B：Sterile supernatant；C：The crude lipopeptides
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图 7 Bacillus amyloliquefaciens E3菌株脂肽粗提物对Dickeya zeae EC1的抑制作用
“*”、“**”分别表示0.05和0.000 1水平的差异显著性(t检验)
Figure 7. Inhibitory effect of crude lipopeptides extracted from Bacillus amyloliquefaciens E3 strain against Dickeya zeae EC1
“*”，“**” indicate significance differences at the levels of 0.05 and 0.000 1 respectively (t test)
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图 8 Bacillus amyloliquefaciens E3菌株的抗真菌谱
A：茄病镰刀菌Fusarium solani；B：黄曲霉Aspergillus flavus；C：黑曲霉Aspergillus niger；D：雪腐镰刀菌Fusarium nival；E：大肠埃希菌Escherichia coli；F：柑橘溃疡菌Xanthomonas citri pv. citri；G：水稻基腐病菌 Dickeya zeae EC1；H：茄科劳尔氏菌Ralstonia solanacearum；I：洋葱伯克氏菌Burkholderia cenocepacia；J：香蕉基腐病菌Dickeya zeae MS1
Figure 8. The antimicrobial spectrum of strain Bacillus amyloliquefaciens E3 strain
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图 9 HPLC 纯化分离抑菌物质各馏分活性检测图
1~8：HPLC收集的馏分；9：脂肽粗提物；10：甲醇
Figure 9. Determination of antibacterial activity of each fraction isolated and purified by HPLC
1−8: Fractions collected by HPLC; 9: Crude lipopeptides; 10: Methanol
表 1 水稻基腐病菌拮抗菌 E3菌株的主要生理生化特性
Table 1 The main physiological and biochemical characteristics of Bacillus amyloliquefaciens E3 strain
测定指标
Detected index生化反应
Biochemical
reaction测定指标
Detected index生化反应
Biochemical
reaction接触酶 Contact enzyme 阳性 Positive 苯丙氨酸脱氢酶 Phenylalanine dehydrogenase 阴性 Negative 氧化酶 Oxidase 阴性 Negative 2%(w) NaCl耐盐试验 Salt tolerance test with 2% NaCl 阳性 Positive 明胶水解 Gelatin hydrolysis 阴性 Negative 5%(w) NaCl耐盐试验 Salt tolerance test with 5% NaCl 阳性 Positive V-P试验 V-P test 阳性 Positive 7%(w) NaCl耐盐试验 Salt tolerance test with 7% NaCl 阳性 Positive 丙酸盐利用 Propionate utilization 阴性 Negative 10%(w) NaCl耐盐试验 Salt tolerance test with 10% NaCl 阳性 Positive 脲酶试验 Urease test 阴性 Negative 淀粉水解 Starch hydrolysis 阳性 Positive 吲哚试验 Indole test 阴性 Negative 葡萄糖产酸 Glucose acidogenesis 阳性 Positive 硝酸盐还原 Nitrate reduction 阳性 Positive 柠檬酸盐利用 Citrate utilization 阳性 Positive 酪氨酸水解 Tyrosine hydrolysis 阴性 Negative 酪素水解 Casein hydrolysis 阳性 Positive pH5.7肉汤 pH5.7 Broth 阳性 Positive pH6.8肉汤 pH6.8 Broth 阳性 Positive 
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