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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 accurately and quickly screen Plenodomus lindquistii of sunflower black-stalked seeds carried by sunflower seeds, the sensitivities of PCR primers 320FOR / 320RVE, LEPB / LEPF and LLF / LLR for three pairs of sunflower black-stalk pathogens were compared by the conventional PCR method. Kazakhstan imported 120 batches of oil sunflower seed samples for PCR detection, and tested positive samples for product sequencing and pathogen isolation. The results showed that the primer 320FOR / 320RVE had the highest detection sensitivity of 10-5ng / μL and the primers LEPB / LEPF and LLF / LLR of 10-4ng / μL and 10-3ng / μL, respectively. The 120 batches of oil sunflower seed samples were detected with primers 320FOR / 320RVE, LEPB / LEPF and LLF / LLR, and the positive detection rates were 65%, 50% and 45% respectively. The positive detection rate and primer detection sensitivity Consistent. The sequence of the product amplified by the positive sample had the highest sequence homology with that of GenBank in sunflower. The colonies of the isolated pathogens were milky white to off-white, the conidia were dark brown, spherical and produced clear conidia, Spore cells, colorless, kidney type, both ends of the oil ball, and the reported description of the pathogen characteristics of sunflower black stem bacteria consistent description of the initial identification of Kazakhstan sunflower sunflower seeds present in the black stalk.