摘要
葡萄座腔菌属(Botryosphaeria)真菌是导致多种水果溃烂的重要致病菌,从形态上很难区分。选取了4个DNA片段(ITS、LSU、SSU、EF-1α)作为3种重要苹果致病菌(Botryosphaeria stevensii、Botryosphaeria dothidea、Botryosphaeria obtusa)的候选条形码进行PCR扩增、Sanger测序和序列分析。结果表明,PCR扩成功率ITS>SSU>LSU=EF-1α,ITS的PCR扩增成功率达到100%;序列分析发现ITS和EF-1α基因种内序列保守,种间差异明显,而且B.dothidea菌种在EF-1α基因中存在一段100 bp左右的特异片段,可以特异区分该菌株,但核糖体基因LSU和SSU种间、种内没有明显差异;结合Gen Bank中的葡萄座腔属真菌ITS序列进行系统发育分析,显示3种葡萄座腔菌属菌株分别位于3个独立的分支,分析结果与形态鉴定结果一致。推荐使用ITS和EF-1α两个基因共同作为这3种葡萄座腔属真菌的条形码。
Species of Botryosphaeria are the important pathogenic fungi on many kinds of fruits. They are very always difficult to distinguish from each other based on morphological characters. In this study, to identificate of three important apple-pathogens Botryosphaeria stevensii, Botryosphaeria dothidea, Botryosphaeria obtuse. 4 gene fragments were selected, which encoded ITS、LSU、SSU、EF-1α,to be the candidate DNA barcodes. By using of PCR amplification, Sanger sequencing and data analysis,we aimed to find the molecular markers in the identification of these species. The results showed that, based on the PCR and sequencing success,it was ranked as follow：ITS 〉SSU 〉LSU =EF-1α,and ITS primer worked best（100%）.According to the acquired sequences,ITS and EF-1α were quite conserved among different strains of the same species,and have the significant differences between the species. Meantime,a region of EF-1α（approx. 100 bp） was exclusively existed in the investigated strains of B. dothidea,which therefore could be used for the distinction of this species. However,the sequences of LSU and SSU have the same variation within and between the species. Combining the available ITS sequences in Gene Bank,we constructed maximum likelihood tree to identify the phylogenetic relationships of Botryosphaeria. The results showed that three investigated species belonged to three monophyletic clades. These results were congruent with the taxonomic units based on the morphological characters. Accordingly, we recommended the usage of ITS and EF-1α to be the efficient molecular makers in the identification of B. stevensii、B. dothidea、B. obtuse.
出处
《湖北农业科学》
2018年第2期126-131,共6页
Hubei Agricultural Sciences
基金
国家质量监督检验检疫总局科技计划项目(2015IK157)
