云杉叶疫病根球壳孢菌的分子检测

Rapid molecular detection of spruce needle blight caused by Rhizosphaera kalkhoffii

从四川二郎山人工云杉林分离得到云杉叶疫病病原菌,采用真菌通用引物ITS1和ITS4扩增得到的序列与NCBI中的根球壳孢菌(Rhizosphaera kalkhoffi)相似度达99%。比较分析了NCBI及四川二郎山分离到的根球壳孢菌ITS区序列,采用软件primer5.0设计20个引物。通过对32株菌株DNA进行PCR扩增及产物测序,筛选得到3个特异性引物对ZYZ6、ZYZ7、ZYZ8。改变DNA模板浓度进行扩增检测,在25μL PCR反应体系中,引物对ZYZ6F/ZYZ6R检测灵敏度最高,可达到0.1 pg/μL。利用ZYZ6F/ZYZ6R对四川二郎山人工云杉针叶DNA进行检测,扩增得到310 bp左右的条带,测序结果与Gen Bank中根球壳孢菌相似度达99%。

Pathogen causing spruce needle blight was isolated from the artificial spruce forest on Erlang Moutain, and the amplification sequence from the pathogen using fungus general primers ITS1 and ITS4 showed 99% similarity with the Rhizosphaera kalkhoffii in NCBI. Internal transcribed spacer（ITS） sequences of the isolated pathogen and Rhizosphaera kalkhoffii（R. kalkhoffii） in NCBI were compared and analyzed, and 20 pairs of primers were designed with softwareprimer5.0. The amplification results for 32 strains combined with sequencing results showed that 3 primer pairs ZYZ6, ZYZ7 and ZYZ8 specific for R. kalkhoffii were obtained. The sensitivity of the 3 specific primers was tested by changing their concentration. The results showed primer pair ZYZ6F/ZYZ6 R had the highest sensitivity which can detect the pathogen at genomic DNA concentration of 0.1 pg/μL in 25 μL PCR reaction system. Primer pair ZYZ6F/ZYZ6 R was used to detect the pathogen from the DNA of the plant leaves of spruce. Sequencing results showed the sequence of the 310 bp amplified bands showed 99% similarity with the recorded sequence of strain Rhizosphaera kalkhoffii in Gen Bank.