甘蔗组培苗2种污染细菌的分离与鉴定

刘红坚 李松 何为中 刘俊仙 刘丽敏 卢曼曼 游建华 黄诚华 林善海

摘  要：细菌污染是甘蔗组培苗生产上的一个主要问题。为明确甘蔗组培中常出现的污染细菌的种类，本研究通过梯度稀释的方法分离污染细菌，结合生化特性、16S rRNA和phaC基因序列比对及系统发育分析鉴定其分类地位。结果表明，共分离获得2种不同类型污染细菌，除了甘露醇，其余9个生化指标完全一致。基因序列比对发现，2种类型分离物的种内2个基因序列完全一致，而种间16S rRNA序列只有2个碱基差异，种间phaC序列有23个碱基差异。2个基因序列构建的系统发育树显示，2种细菌遗传距离较近，并均能形成独立的2个分支。结合生化特征、序列比对及系统发育树，将2种污染细菌分别鉴定为巨大芽孢杆菌（Bacillus megaterium）和阿氏芽孢杆菌（B. aryabhattai），且首次报道阿氏芽孢杆菌为甘蔗组织培养中的污染细菌。本研究揭示了巨大芽孢杆菌可能来源于甘蔗的内生菌，而阿氏芽孢杆菌来源于环境。

关键词：甘蔗;组培苗;污染细菌;芽孢杆菌;系统发育树

Abstract： Contamination is a main issue in sugarcane tissue culture. The purpose of this study was to determine the contaminative bacteria frequently occurred during the production of sugarcane tissue culture. The gradient dilution method was used to isolate bacteria， and the toxonomic position was identified combining the biochemical， sequence alignment of 16S rDNA and phaC genes， phylogeomic analysis. The results showed that two type contaminative bacteria were obtained from the sugarcane tissue culture nutrient solution， and nine tested biochemical characters were the same between the two bacteria except mannitol utilization. Sequence alignment showed that intraspecific sequences of 16S rDNA and phaC genes were completely uniformity respectively in the two bacteria， but the difference of the two nucleotide bases in 16S rDNA and 23 in phaC sequences existed between the two bacteria. The phylogenic tree constructed based on 16S rDNA and phaC genes sequences respectively also indicated that the two bacteria were closest each other and clustered into two absolutely independent branches. Combining the biochemical characters， sequence alignment and phylogenetic analysis， the two contaminative bacteria were identified as Bacillus megaterium and B. aryabhattai， respectively. This is the first report of B. aryabhattai as the contaminative bacterium in sugarcane tissue culture. This study revealed that B. megaterium probably came from endophyte and B. aryabhattai from environment.

Keywords： sugarcane; tissue culture seedling; contaminative bacterium; Bacillus; phylogenetic tree

甘蔗（Saccharum officinarum L.）是热带和亚热带气候种植的重要经济作物之一，占全球产糖量的80%左右。甘蔗在广西农业、工业作物中占据了极其重要的位置。许多工业和工厂都依赖于甘蔗及其副产品提供的原材料。它被认为是将太阳能转化为有价值且可应用的化学能的主要作物之一，如蔗糖和生物质。甘蔗主要通过种茎进行无性繁殖，但长年无性繁殖容易引起线虫、真菌、细菌和病毒等引起的系统性病害，导致种性退化、产量损失[1-2]。……