一株甘蔗黑穗病菌的分离与系统发育分析

Isolation and Systemic Development Analysis of a Strain of Ustilago scitaminea

甘蔗黑穗病是由黑粉菌(Ustilago scitaminea Syd.)引起的一种世界性病害,严重危害甘蔗的生产,对该病病原菌的研究有助于甘蔗抗黑穗病育种。由于rDNA序列兼具保守区域和进化水平不同所致可变区,因此,rDNA序列分析是研究真菌系统发育、分类鉴定和分子检测非常有效的手段。笔者采用单孢分离方法,从高抗品种NCo376的病株上分离单孢,培养菌丝体,提取基因组DNA,分析其rD-NA序列,以期丰富该真菌的遗传多样性研究。用真菌rDNA序列分析的通用引物ITS1和ITS4,通过ITS-PCR得到目的片段,克隆在pMD18-T载体上后进行测序,结果显示该片段的长度为692bp,与Genebank中已报道的真菌序列进行Blast和系统发育树分析,表明所获得的序列与Sporisorium属真菌具有更高的亲缘关系,而与Ustilago属真菌的亲缘关系较远,这与一直沿用的甘蔗黑穗病菌的命名似乎并不吻合,有待进行更多的研究以证实。

Sugarcane smut caused by Ustilago scitaminea is a worldwide disease and causes serious damage to sugarcane. Research on the pathogen of sugarcane smut is aid to sugarcane breeding resistant to smut. With the conservative regions and variable regions in rDNA gene sequences, the analysis of rDNA sequences is very efficient in research of fungi systemic development, classification identification and molecular detection. The method of single spore isolation is occupied in this experiments. Single spore was isolated from smut whip in infected plant of smut high resistant sugarcane variety of NCo376, and then its mycelia were prepared and used for isolation of genomic DNA. The rDNA analysis of the fungus will increase understanding of it. The universal primers of ITS1 and ITS4 for fungi were used in amplification of genomic rDNA sequences. A target fragment was obtained by ITS-PCR. Then, the fragment is cloned into the vector of pMD18-T before sequencing. The result of sequencing showed that the fragment is 692 bp in length. The results of Blast in Genbank and the analysis of fungal rDNA systemic development show that the relation between this fragment to the fungi of Sporisorium is closest, while the relation between it to the fungi of Ustilago is much far. It suggests that the name of Ustilago scitaminea for sugarcane smut isn＇t consistent to the result of rDNA systemic development analysis. It still needs more experiments to support this hypothesis.