油菜花期东方蜜蜂及西方蜜蜂蜜囊细菌多样性

Diversity of bacteria in the honey stomach of Apis cerana and Apis mellifera during the rape blooming period

【目的】本研究对油菜花期的东方蜜蜂Apis cerana、西方蜜蜂Apis mellifera蜜囊细菌进行分离培养及分子鉴定,通过序列比对及系统发育树分析彼此间的多样性差异和来源,为蜂群饲养管理过程中细菌的控制和应用提供理论依据。【方法】对东、西方蜜蜂蜜囊细菌进行分离、纯化培养;并对分离菌株的16S r DNA基因片段进行扩增和测序,序列比对后进行系统发育分析。【结果】蜜囊中解淀粉芽孢杆菌Bacillus amyloliquefaciens占总菌数的百分比为69.81%-95.97%,P值在0.007-0.009之间,显著高于其它细菌占总菌数百分比（4.03%-30.19%）,是优势菌;275株培养特征和表现型不同的菌株共有9种不同的单倍型菌株,通过比对其相似度〉99%;其中16株细菌16S r DNA基因序列已提交Gen Bank数据库,登录号：kf710012-kf710033;东方蜜蜂蜜囊细菌包括2个属中的5个种,西方蜜蜂蜜囊细菌包括5个属中的7个种,花蜜中细菌包括2个属中的2个种;系统发育树分析表明,簇Ⅰ和簇Ⅱ分别与参考菌株EF565944、EF565939和EF565936属同一种;未分离到参考簇Ⅴ;簇Ⅲ、簇Ⅳ、簇Ⅵ、簇Ⅶ这4个属的蜜囊细菌是首次报道并提交Gen Bank。【结论】东、西方蜜蜂蜜囊细菌除了共有的优势菌解淀粉芽孢杆菌,细菌多样性有明显的差异;与国外蜜蜂蜜囊研究存在地理差异;解淀粉芽孢杆菌可以作为蜂粮的添加剂,提高蜜蜂抗病性。

[Objectives] To analyze the diversity, origin and differences in the cultured bacterial community present in the honey stomach ofApis cerana andApis mellifera. [Methodsl Bacteria were isolated and cultivated to obtain pure isolates in the honey stomach of A.cerana and A. mellifera. The isolates were compared and a phylogeny constructed analysis based on 16S rDNA sequences amplification and sequencing. [Results[ Bacillus amyloliquefaciens was the dominant bacteria comprising 69.81% to 95.97% of the total found in each bee species respectively, significantly higher than other bacteria, which comprised just 4.03% to 30.19%. P-values for these differences were 0.007 to 0.009, respectively. A total of 275 bacterial isolates were selected and identified from different phenotypes. All bacterial 16S rRNA sequences from the different samples showed nine haplotypes and 〉99% similarity to the phylotype in GenBank. 16 isolates were submitted to GenBank and obtained accession numbers from kf710012 to kf710033. The honey stomach ofA. cerana contained five bacterial species from two genera, in comparison, the honey stomach of A. mellifera contained seven species from five genera and nectar contained two species from two genera. Phylogenetic analysis indicated that clusters I and 1I were the same species as the previously described EF565944, EF565939 and EF565936. Cluster V （Lactobacillus） was not found in this study. Clusters III, IV, VI and VII were also not found in the reference strains. This is the first time that bacteria in the honey stomach of A. cerana and A. mellifera have been described and submitted to GenBank. [Conclusion] Except for the common dominant bacteria B. amyloliquefaciens, bacterial diversity in the honey stomach is obviously different between A. cerana and A. mellifera. Comparison with previous, overseas studies of the honey stomach bacterial community suggests that there are geographic differences in bacterial community composition. B. amyloliquefaciens can be used as a food additive that enhances disease resistance in honeybees.