| 烟草黑胫病拮抗细菌的分离、鉴定及发酵条件优化 |
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| 引用本文： | 何明川,施春兰,魏聪聪,曾舒泉,兰明先,唐萍,王志江,伍显录,陆光钰,谢永辉.烟草黑胫病拮抗细菌的分离、鉴定及发酵条件优化[J].南方农业学报,2022,53(6):1604-1615. |
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| 作者姓名： | 何明川 施春兰 魏聪聪 曾舒泉 兰明先 唐萍 王志江 伍显录 陆光钰 谢永辉 |
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| 作者单位： | 1. 云南农业大学植物保护学院, 云南昆明 650201;2. 云南省烟草公司昆明市公司, 云南昆明 650051;3. 红塔烟草 (集团) 有限责任公司, 云南玉溪 653100 |
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| 基金项目： | 云南省科技计划项目(202105AC160037)云南省烟草公司科技计划项目(2020530000242026) |
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| 摘 要： | 【目的】从美洲大蠊肠道筛选获得对烟草黑胫病病原菌具有拮抗作用的拮抗菌株，明确拮抗菌株的分类地位、优化其发酵条件及测定其对烟草黑胫病的室内防治效果，为烟草黑胫病的生物防治提供菌种资源。【方法】以美洲大蠊肠道为材料，利用稀释涂布平板法及平板对峙法筛选对烟草黑胫病具有拮抗作用的菌株，根据形态特征、生理生化、16S rDNA和gyrB基因序列分析对拮抗菌株进行鉴定；以细菌发酵液的OD600为指标，对拮抗菌株发酵培养基及发酵条件进行单因素及正交试验；通过室内盆栽试验测定拮抗菌株对烟草黑胫病的防治效果。【结果】采用稀释涂布平板法从美洲大蠊肠道分离得到200株细菌，经平板对峙法筛选得到对烟草黑胫病具有较强拮抗作用的菌株MC2-1，其抑菌带宽为8.00 mm，平均抑菌率为62.87%。结合菌落形态、生理生化及16S rDNA和gyrB系统发育进化树分析结果，鉴定菌株MC2-1为贝莱斯芽孢杆菌（Bacillus velezensis）；其最适培养基配方为:牛肉浸膏8 g/L，酵母浸粉5 g/L，麦芽糖10 g/L；最佳培养条件为:接菌量10%、装液量30 mL、培养基初始pH 7、转速180 r/min、培养温度36℃、培养时间60 h；优化后的培养基和发酵条件菌液浓度（OD600分别为2.72和2.81）均显著高于原始培养基和发酵条件的菌液浓度（OD600分别为2.31和2.56）（P<0.05）；盆栽试验结果显示，优化后菌株MC2-1对烟草黑胫病的平均防效为63.92%，显著高于优化前的53.72%。【结论】优化后的方案可提高菌株MC2-1的菌体量，并增强对烟草黑胫病的防治效果。菌株MC2-1具有开发成为烟草黑胫病生防菌的潜力。
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| 关 键 词： | 美洲大蠊 烟草黑胫病 拮抗细菌 发酵条件 生物防治 |
| 收稿时间： | 2022-01-06 |
Isolation,identification and optimization of fermentation conditions of antagonistic bacteria against tobacco black shank |
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| HE Ming-chuan,SHI Chun-lan,WEI Cong-cong,ZENG Shu-quan,LAN Ming-xian,TANG Ping,WANG Zhi-jiang,WU Xian-lu,LU Guang-yu,XIE Yong-hui.Isolation,identification and optimization of fermentation conditions of antagonistic bacteria against tobacco black shank[J].Journal of Southern Agriculture,2022,53(6):1604-1615. |
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| Authors: | HE Ming-chuan SHI Chun-lan WEI Cong-cong ZENG Shu-quan LAN Ming-xian TANG Ping WANG Zhi-jiang WU Xian-lu LU Guang-yu XIE Yong-hui |
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| Affiliation: | 1. College of Plant Protection, Yunnan Agricultural University, Kunming, Yunnan 650201, China;2. Kunming Branch, Yunnan Tobacco Company, Kunming, Yunnan 650051, China;3. Hongta Tobacco(Group)Co., Ltd, Yuxi, Yunnan 653100, China |
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| Abstract: | 【Objective】 To clarify classification of bacterial strain with antagonism effect on tobacco black shank from the intestinal tract of Periplaneta americana，to optimize fermentation conditions and to determine indoor control effects of tobacco black shank，so as to provide bacterial resources for biological control of tobacco black shank.【Method】 Intestinal tract of P.americana was used as material，and the antagonistic strains against tobacco black shank were screened by dilution coating plate method and plate confrontation method.The strains were identified according to their morphological characteristics，physiological and biochemical characteristics，16S rDNA and gyrB gene sequence analysis.Single factor and orthogonal test were carried out on the fermentation medium and fermentation conditions of bacterial fermentation broth with OD600 value as the index.Control effect of the strain on tobacco black shank was determined by indoor pot experiment.【Result】 200 bacterial strains were isolated from the intestinal tract of P.americana by dilution coating method.A strain MC2-1 screened by plate confrontation method，with an average inhibitory rate of 62.87% and an antibacterial bandwidth of 8.00 mm，was strong antagonistic against tobacco black shank.Considering the results of colony morphology，physiological and biochemical detection，and phylogenetic evolutionary tree of 16S rDNA and gyrB，the strain MC2-1 was identified as Bacillus velezensis.The optimum medium formula was:beef extract 8 g/L，yeast extract 5 g/L and maltose 10 g/L；the optimum culture condition was:inoculation amount 10%，liquid volume 30 mL，initial pH value of medium 7，rotation speed 180 r/min，culture temperature 36℃，culture time 60 h.The bacterial solution concentration of optimized culture medium and fermentation condition（OD600 were 2.72 and 2.81，respectively）were significantly higher than that the original culture medium and fermentation condition（OD600 were 2.31 and 2.56，respectively） （P<0.05）.The results of pot experiment showed that the average control effect of the optimized strain MC2-1 on tobacco black shank was 63.92%，was significantly higher than the 53.72% before the optimization.【Conclusion】The optimized scheme can increase the bacterial mass of MC2-1 and enhance the control effect of tobacco black shank.Strain MC2-1 is potential to be developed as a biocontrol bacterium against tobacco black shank. |
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