用传统分离培养法和高通量测序技术分析印度块菌子囊果内细菌的群落结构

Community composition of bacteria associated with ascocarps of Tuber indicum using traditional culture method and Roche 454 high-throughput sequencing

块菌是重要的经济真菌,在其生长发育过程中,细菌扮演了重要角色。本文利用传统分离培养方法和高通量测序技术分析了印度块菌(Tuber indicum)子囊果内细菌的群落结构。共分离得到细菌532株,根据物种累积曲线,选取其中的112株细菌进行了16SrRNA基因序列分析,共鉴定出4属40种,其中假单胞菌属(Pseudomonas)菌株占所测菌株数的80%,不动杆菌属(Acinetobacter)占12.5%,链霉菌属(Streptomyces)占5%,贪噬菌属(Variovorax)占2.5%。通过对印度块菌子囊果16Sr RNA基因的V1–V3区高通量测序分析,共获得细菌序列9,862条,分属于7门43属220种,其中变形菌门、拟杆菌门和放线菌门的物种占总物种数的99.7%,是印度块菌子囊果内的优势细菌。黄杆菌属(Flavobacterium)、壤霉菌属(Agromyces)、微杆菌属(Microbacterium)、剑菌属(Ensifer)和寡养食单胞菌属(Stenotrophomonas)的物种数占总物种的86.3%,是印度块菌子囊果内细菌的优势属。研究结果表明,采用胰蛋白大豆培养基仅分离得到印度块菌子囊果内少数细菌物种,而采用高通量测序技术分析发现,印度块菌子囊果内细菌物种种类丰富,群落结构复杂。

Truffles are important economic fungi,and bacteria play an important role in their growth and development. We investigated the bacterial community composition inside ascocarps of Tuber indicum. Using the traditional culture method,532 isolates were obtained from ascocarps. Using the rarefaction curve,the 16S rRNA gene was collected and sequenced from 112 purified isolates to identify 4 genera and 40 species. Isolates of the genera Pseudomonas,Acinetobacter,Streptomyces and Variovorax accounted for 80%,12.5%,5% and 2.5% of the number of these sequenced isolates,respectively. On the other hand,9,862 sequences of the bacterial V1-V3 region of 16S rRNA gene,which represented 220 species,were analyzed via Roche 454 high-throughput sequencing. Species of the phyla Proteobacteria,Bacteroidetes,and Actinobacteria were dominant,accounting for 99.7% of all identified species. Genera Flavobacterium,Agromyces,Microbacterium,Ensifer,and Stenotrophomonas were dominant among the bacteria identified with this alternative method,accounting for 86.3% of the number of total species. It was found that relatively few bacterial species were isolated from ascocarps of T. indicum when analyzed via traditional culture method. The bacterial population associated with ascocarps of T. indicum was augmented when analyzed by Roche 454 high-throughput sequencing,which indicates that this latter method provides more comprehensive results.