不同引物PCR检测向日葵黑茎病菌的灵敏度比较及应用

Comparison of sensitivity and application of different primer pairs for the detection of Plenodomus lindquistii

为准确快速筛查进境油葵种子中携带的向日葵黑茎病菌Plenodomus lindquistii,采用常规PCR方法比较了3对向日葵黑茎病菌PCR检测引物320FOR/320RVE、LEPB/LEPF和LLF/LLR的灵敏度,对哈萨克斯坦进境120批油葵种子样品进行PCR检测,并对检测为阳性的样品进行产物序列测定及病原菌分离。结果显示,引物320FOR/320RVE的检测灵敏度最高,可达10-5ng/μL,引物LEPB/LEPF和LLF/LLR分别为10-4ng/μL和10-3ng/μL。利用引物320FOR/320RVE、LEPB/LEPF和LLF/LLR对120批进境油葵种子样品进行检测,阳性检出率分别为65%、50%和45%,阳性检出率高低与引物的检测灵敏度相一致。阳性样品扩增的产物序列与Gen Bank中向日葵黑茎病菌的序列同源性最高;分离的病原菌菌落为乳白色至灰白色,分生孢子器黑褐色、球形并产生透明状分生孢子液,分生孢子单胞、无色、肾型、两端有油球,与已报道的向日葵黑茎病菌的病原特征描述一致,初步鉴定哈萨克斯坦进境油葵种子中存在向日葵黑茎病菌。

In order to detect Plenodomus lindquistii in imported sunflower seeds precisely and rapidly,the detection sensitivities of three pairs of PCR primers 320 FOR /320 RVE,LEPB / LEPF and LLF / LLR for P. lindquistii were compared,and the sequences of PCR positive products were analyzed by screening120 sunflower seeds from Kazakhstan and isolated strains from the samples in this study. The results showed that the hyphal DNA with concentration of 10- 5,10- 3,10- 4ng / μL was detected by using primers 320 FOR /320 RVE, LEPB / LEPF and LLF / LLR, respectively, which showed the detection sensitivity of 320 FOR /320 RVE was the highest. Screening 120 samples of sunflower seeds with the primers 320 FOR /320 RVE,LEPB / LEPF and LLF / LLR demonstrated that 65%,50% and 45% of samples were positive samples,respectively. It indicated that the detection rate of positive samples was consistent with the primer sensitivity. The sequences of positive sample amplification product shared the highest homology with P. lindquistii sequences in Gen Bank. The colonies were milk white to light grey color; pycnidia were dark brown,sphericald and produced hyaline liquid,which contained hyaline pycnidiospores,unicellular,kidney-shaped,with one guttulate at each end. Moreover,the sequence analysis and morphological feature of the strains separated from the samples indicted that P. lindquistii was imported from Kazakhstan in the sunflower seeds.