草地贪夜蛾幼虫肠道细菌的分离鉴定及纤维素降解细菌的筛选
Isolation and identification of the intestinal bacteria, and screening of the cellulolytic bacteria, of Spodoptera frugiperda (Lepidoptera: Nocturid) larvae
李宏伟；杨晓洁；向奕舟；林连兵；张棋麟
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DOI：10.7679/j.issn.2095-1353.2020.061
作者单位：昆明理工大学生命科学与技术学院，昆明 650500；云南省高校饲用抗生素替代技术工程研究中心，昆明 650500
中文关键词：草地贪夜蛾幼虫；可培养肠道细菌；菌种鉴定；纤维素降解；防治靶标
英文关键词：Spodoptera frugiperda larvae; culturable intestinal bacteria; identification of bacteria species; cellulose degradation; control targets
中文摘要：
【目的】 本研究旨在明确草地贪夜蛾Spodoptera frugiperda幼虫肠道可培养细菌组成，筛选纤维素降解细菌。【方法】 采用传统细菌培养及16S rDNA分子标记相结合的方法分离鉴定草地贪夜蛾幼虫肠道可培养细菌；采用刚果红染色法筛选纤维素降解细菌，并通过3，5-二硝基水杨酸（DNS）法测定不同pH（5.0-9.0）条件下的纤维素酶活力。【结果】 从草地贪夜蛾幼虫中筛选分离出14种肠道细菌菌株，分别隶属放线菌门（Actinobacteria）、厚壁菌门（Firmicutes）、变形菌门（Proteobacteria）等3门11属，即谷氨酸棒杆菌属（Glutamicibacter）、肠球菌属（Enterococcus）、芽胞杆菌属（Bacillus）、葡萄球菌属（Staphylococcus）、摩根菌属（Morganella）、肠杆菌属（Enterobacter）、志贺氏菌属（Shigella）、克洛诺杆菌属(Cronobacter)、克雷伯氏菌属（Klebsiella）、沙雷氏菌属（Serratia）、苍白杆菌属（Ochrobactrum）。2株产纤维素酶细菌隶属厚壁菌门芽胞杆菌属。酶活力测定结果显示：2株纤维素降解细菌的纤维素酶在pH 6.0-8.0均有相对较高的活性，在pH 8.0时，纤维素酶活最高。【结论】 草地贪夜蛾幼虫肠道中细菌种类多样，其肠道内存在纤维素降解细菌。在偏碱性条件下，纤维素降解细菌的纤维素酶活力显著高于酸性条件。研究结果丰富了纤维素降解细菌资源，在饲料生产、食品加工、化学能源等方面具有应用前景。此外，草地贪夜蛾作为重大农业害虫，其肠道有益菌群，有望成为新的防治靶标。
英文摘要：
[Objectives] To determine the composition of culturable bacteria, and screen cellulolytic bacteria, in the intestine of Spodoptera frugiperda larvae. [Methods] The traditional bacterial culture method and the 16S rDNA molecular marker were used to identify culturable bacteria in the intestine of S. frugiperda larvae. Congo red staining was used to screen cellulolytic bacteria, and cellulase activity was determined with the 3,5-dinitrosalicylic acid (DNS) method under different pH (5.0-9.0). [Results] Fourteen intestinal bacterial strains were isolated, including Glutamicibacter, Enterococcus, Bacillus, Staphylococcus, Morganella, Enterobacter, Shigella, Cronobacter, Klebsiella, Serratia and Ochrobactrum, which belong to eleven genera of three phyla; Actinobacteria, Firmicutes and Proteobacteria. Among these, two cellulolytic bacteria strains from the Bacillus genus of Firmicutes were identified. The results show that the cellulase activities of these two cellulolytic strains were relatively higher under pH 5.0-8.0 and peaked at pH 8.0. [Conclusion] The intestine of S. frugiperda larvae contains a diversity of bacteria. The cellulase activity of cellulolytic strains was significantly higher under alkaline than under acid conditions. These results provide new information on cellulolytic bacteria, and contribute to their use in feed production, food processing and chemical energy. In addition, beneficial intestinal bacteria are expected to become a new target for controlling S. frugiperda which is now a major global pest.