Screening, Identification, and Preliminary Analysis on Antagonistic Bacillus sp. against Potato Dry Rot
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摘要:
目的 探究分离自青稞白酒糟醅的菌株对马铃薯干腐病的生防效果，为马铃薯干腐病的生物防控提供初步的理论依据。 方法 采用平板对峙法，测定9株分离自青稞白酒糟醅的菌株对4株马铃薯干腐病病原真菌的抑菌活性。通过形态学、生理生化及分子生物学方法对活性菌株进行鉴定。采用平板透明圈法和排油圈法，分别测定活性菌株发酵液分泌抑菌蛋白和产生脂肽类物质的能力。采用牛津杯法，以马铃薯干腐病病原菌为指示菌，测定活性菌株发酵液有机溶剂萃取物的抑菌活性及其在不同条件下抑菌活性的稳定性。 结果 （1）筛选到2株对马铃薯干腐病菌具有较强抑制活性的菌株JZ3-4-7和JZ3-1-15，其中JZ3-1-15抑菌活性稳定。（2）经鉴定，JZ3-1-15为贝莱斯芽孢杆菌（Bacillus velezensis）。（3）JZ3-1-15的发酵液能产生蛋白酶、淀粉酶和纤维素酶，并且能够产生脂肽类物质。（4）JZ3-1-15发酵液的正丁醇萃取物对马铃薯干腐病病原真菌青9D-2-6的抑菌活性最高，抑制率达80.07%。（5）JZ3-1-15发酵液的正丁醇萃取物可耐121 ℃高温，热稳定性较强；具有较高的酸碱稳定性，pH值为8时抑菌活性达到最高；紫外线照射处理对抑菌活性无影响。 结论 JZ3-1-15及其正丁醇萃取物对马铃薯干腐病菌具有强抑菌活性，有望将其开发为马铃薯新型生防保鲜菌剂，并从中分离到对马铃薯干腐病菌具有新颖作用靶点的活性化合物及先导化合物。 Abstract:Objective Bacillus sp. isolated from Hordeum vulgare L. distiller’s grain were explored as a potential biocontrol agent against the potato dry rot disease. Method The plate confrontation method was applied to examine the antagonistic activities of 9 Bacillus strains isolated from the barley wine distiller’s grain against 4 potato dry rot pathogens. Active strains were then identified by morphological, physiological, biochemical, and molecular biology methods. The plate transparent ring and discharge of oil ring tests were used to determine the capacity of the selected strains in secreting bacteriostatic proteins and lipopeptides. Using the potato dry rot pathogens as indicator in the Oxford cup test, the antibacterial activity and the stability under different conditions of the extracts of the fermentation broths of the active strains were determined. Result (1) The Bacillus sp., JZ3-4-7 and JZ3-1-15 displayed significant inhibitory activities against the potato dry rot pathogens in the screening. JZ3-1-15 also exhibited a desirable stability. (2) JZ3-1-15 was subsequently identified as B. velezensis. (3) The JZ3-1-15 fermentation broth contained protease, amylase, cellulase, as well as lipopeptides. (4) The n-butanol extract of JZ3-1-15 fermentation broth had a significant inhibitory rate of 80.07% against the pathogen, Qing 9D-2-6. (5) The same extract withstood a temperature as high as 121 ℃ maintaining a strong thermal stability. It was also stable under acidic and alkaline conditions with a peak anti-pathogenicity at pH 8, and its activity not affected by ultraviolet. Conclusion JZ3-1-15 and the n-butanol extract of its fermentation broth showed significant inhibitory activity on the potato dry rot pathogens. It appeared that they could be developed as a new biocontrol agent against the target pathogens and/or a preservative for fresh potatoes. The effective compounds or precursors might also be isolated from them for combating the potato dry rot disease and further studies. -
图 1 菌株JZ3-1-15的培养性状及革兰氏染色特征
注：A为培养24 h后的菌落形态；B为培养72 h后的菌落形态；C为革兰氏染色结果。
Figure 1. Culture and gram-staining characteristics of JZ3-1-15
Note: A: colony morphology after 24 h plate culture. B: colony morphology after 72 h plate culture. C: gram stained bacteria.
图 2 基于16S rDNA序列构建的菌株JZ3-1-15系统发育分析
Figure 2. Phylogenic analysis on JZ3-1-15 based on 16S rDNA sequence
图 3 菌株JZ3-1-15发酵液生防因子分析
注：A为酪蛋白培养基；B为淀粉培养基；C为羧甲基纤维素培养基；D为葡聚糖培养基；E为排油圈。
Figure 3. Biocontrol factors of JZ3-1-15 fermentation broth
Note: A: casein medium. B: starch medium. C: carboxymethyl cellulose medium. D: glucanase medium. E: discharge of oil ring.
图 4 温度（A）、pH值（B）和紫外线（C）对JZ3-1-15发酵液萃取物的抑菌活性影响
Figure 4. Effects of temperature (A), pH (B), and uv (C) on antibacterial activity of crude JZ3-1-15 fermentation broth extract
表 1 分离自青稞白酒糟醅的菌株抑制马铃薯干腐病病原真菌的活性
Table 1. Inhibitory activity of strains from distiller's grain against pathogens of potato dry rot
菌株
strain青9D-2-6
Qing 9D-2-665D-5-2 青9B-4-6
Qing 9B-4-665C-4-3 病原菌菌落直径
Diameter of
pathogen colony/cm抑制率
Inhibition rate/%病原菌菌落直径
Diameter of
pathogen colony/cm抑制率
Inhibition rate/%病原菌菌落直径
Diameter of
pathogen colony/cm抑制率
Inhibition rate/%病原菌菌落直径
Diameter of
pathogen colony/cm抑制率
Inhibition rate/%JZ3-1-15 3.69±1.23 57.24 4.36±1.47 49.83 4.13±1.45 51.92 4.87±1.37 50.18 JZ3-4-7 4.58±1.64 46.93 4.87±1.72 43.96 5.14±1.21 47.15 5.45±1.72 41.07 JZ3-5-1 7.65±1.22 11.36 8.46±1.32 2.65 7.16±1.32 10.83 6.87±1.56 8.17 JZ3-1-10 7.74±1.36 10.31 8.32±1.26 4.26 8.21±1.65 4.42 6.98±1.66 16.22 JZ3-1-23 7.78±1.47 9.85 7.90±1.02 9.09 7.96±1.85 7.33 6.64±1.31 10.85 JZ3-1-14 7.92±1.58 8.23 8.26±1.59 4.95 7.95±1.49 7.45 7.25±1.45 15.40 JZ3-2-2 8.03±1.29 6.95 8.12±1.55 6.56 8.13±1.72 5.36 6.84±1.35 8.52 JZ2-1-6 8.16±1.34 5.45 7.69±1.83 11.51 7.69±1.57 10.48 7.93±1.57 7.47 JZ3-1-4 8.18±1.51 5.21 7.99±1.35 8.06 8.07±1.58 6.05 7.34±1.52 14.35 CK 8.63±0.64 8.69±1.37 8.59±0.96 8.57±1.23 注：表中数值为平均值±标准差。
Note: The values in the table are mean ± standard deviation.
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表 2 供试菌株对马铃薯干腐病病原真菌抑菌活性的稳定性
Table 2. Stability of antimicrobial activity of strains from distiller's grains against pathogens of potato dry rot
菌株
strain抑菌带缩小率
Inhibition zone
reduction rate/%稳定性评价
Stability evaluationJZ3-1-15 0.78 a ++ JZ3-4-7 25.75 b + 注：表中同一列中不同字母表示处理间差异显著（P＜0.05）。
Note: Different letters in the same column in the table indicate significant differences between the treatments（P＜0.05）.
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表 3 菌株JZ3-1-15的生理生化特征
Table 3. Physiological and biochemical characteristics of JZ3-1-15
试验项目
Pilot projects结果
result试验项目
Pilot projects结果
result柠檬酸利用 Citric acid utilization − 甲基红 M.R − 接触酶 Contact enzyme + V-P − 葡萄糖发酵 Glucose fermentation F ONPG + 蔗糖利用 Sucrose Utilization + 硝酸盐还原 Nitrate reduction + 甘露醇利用 Mannitol utilization + 明胶液化 Gelatin Liquefaction + 麦芽糖利用 Maltose utilization + 石蕊牛乳试验 Litmus Milk Test 凝固 freezing 注：“+”表示阳性，“−”表示阴性。
Note: "+" means positive, "−" means negative.
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表 4 菌株JZ3-1-15发酵液萃取物对病原真菌青9D-2-6的抑菌活性
Table 4. Inhibitory activity of crude extract from JZ3-1-15 fermentation broth against Qing 9D-2-6
萃取物溶液
Extract solution质量浓度
Concentration/（mg·mL−1）抑制率
Inhibition rate/%毒力回归方程
Virulence regression equation回归系数
rEC50/（mg·mL−1） 乙酸乙酯 Ethyl acetate 1.25 20.74±1.38 c y=3.93+0.93 x 0.93 14.06 2.50 23.16±0.67 c 5.00 25.00±1.56 c 10.00 42.17.±1.79 b 20.00 62.67±1.73 a 氯仿 Chloroform 1.25 24.19±1.67 d y=4.05+1.00 x 0.96 8.88 2.50 26.15±1.35 c 5.00 33.64±1.34 c 10.00 52.42±1.37 b 20.00 67.51±2.26 a 正丁醇 N-butanol 1.25 27.76±1.25 d y=4.13+1.22 x 0.98 5.18 2.50 29.84±2.10 d 5.00 37.67±1.63 c 10.00 61.41±1.67 b 20.00 80.07±0.81 a 注：表中同一萃取物的同一列中无相同字母，表示处理间差异显著（P＜0.05）。
Note: Different letters in the same column in the table indicate significant differences between the treatments（P＜0.05）.
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