两株对虾育苗用益生芽孢杆菌的筛选和鉴定
SCREENING AND IDENTIFICATION OF TWO PROBIOTIC BACILLUS STRA
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摘要: 从土壤和虾池中分离到22株具有不同特征的芽孢杆菌,分别将各菌株对数期约5×108个细胞添加到500mL预先加有凡纳滨对虾(Litopenaeus vannamei)无节Ⅲ期幼体的水体中,各菌株在初始水体中含量均约为106个细胞/mL,根据各菌株对无节Ⅲ期到蚤状Ⅱ期幼体变态成活率的影响,从中初步筛选出10株对幼体变态成活有极显著(p<0.01)效果的芽孢杆菌。进一步又从初筛到的10个菌株中复筛到2株对幼体变态成活最为明显的芽孢杆菌,菌株zou4和zou8。两菌株与复筛的其他8株芽孢杆菌相比,均显著(p<0.05)促进幼体变态存活率。根据形态和生理生化特征,菌株zou4和zou8分别初步鉴定为坚强芽孢杆菌(Bacillus firmus)和蜡状芽孢杆菌(B.cereus)。为进一步确定zou4和zou8的分类地位,测定了两菌株的16S rRNA基因序列,分析了相关细菌16S rDNA序列的同源性,构建了系统发育树。结果表明菌株zou4和zou8分别与坚强芽孢杆菌和蜡状芽孢杆菌的同源性最高,相似值均为99%以上。系统发育树上zou4和zou8也分别与坚强芽孢杆菌和蜡状芽孢杆菌聚为一类。综合上述结果,芽孢杆菌zou4和zou8菌株分别被鉴定为坚强芽孢杆菌和蜡状芽孢杆菌。Abstract: 22 strains of Bacillus with different characters were isolated from soil and shrimp ponds. Approximately 5 ×108 logarithmic phase cells of every strain were administered into 500 mL seawater culturing Liptopenaeus vannamei nauplius m larvae respectively. Based on the effect of Bacillus on the survival rate of L. vannamei larvae metamorphosis from nauplius m to zoea Ⅱ, 10 strains which had very significant (p<0.01 ) effects on larval metamorphosis were screened initially. Then from the 10 strains screened, two strains, zou4 and zou8 which had more significant (p<0.05) effects on larval metamorphosis than other strains were selected as potential probiotic Bacillus for L. vannamei larvicuhure. Combining phenotypic and physiological characteristics, zou4 and zou8 were identified initially as B. firmus and B. cereus respectively. To investigate the phylogenetic posi-tion of strains zou4 and zou8, about 1.5kb 16S rRNA genes were sequenced and compared with that of related strains, which re-vealed that strains zou4 and zou8 exhibited the highest levels of similarity to B. firmus and B. cereus respectively, similarity value were all above 99 %. Phylogenetic tree was constructed based on genetic distance analysis, which also showed that strains zou4 and zou8 clustered as B. firmus and B. cereus respectively. Based on the results obtained, strains zou4 and zou8 were identified as B. firmus and B. cereus respectively.
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Keywords:
- Probiotic /
- Bacillus /
- Screening /
- Identification /
- Litopenaeus vannamei /
- Larvae
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