基于流式细胞术的黄瓜霜霉病菌孢子囊计数研究

 为病原菌时空流行动态监测及病害预测模型构建提供病原菌快速计数方法,以卵菌中代表——葫芦科霜霉病病原菌古巴假霜霉菌(Pseudoperonospora cubensis)为例,研究了孢子囊悬浮液的荧光染色方法和基于流式细胞术计数方法。研究表明EDTA和SYBR Green Ⅰ 的联合作用能够对孢子囊进行荧光标记。对SYBR Green Ⅰ 染色剂的最佳染色条件进行研究,结果表明在染色时间相同时,荧光染色剂浓度越高,染色效果越好;同一浓度荧光染色剂,随着染色时间的增加,孢子囊染色率增加。使用流式细胞术对荧光染色的孢子囊进行计数,并与显微镜血球计数板计数结果进行比较,结果显示:流式细胞仪计数结果与显微镜血球计数板计数结果得到的孢子囊浓度没有显著差异(P<0.01),对两种方法获得的孢子囊浓度取对数后进行相关性分析,显示流式细胞仪测定的孢子囊浓度与显微镜血球计数板计数得到的孢子囊浓度高度相关,相关系数为 0.993 4,表明流式细胞术可以应用于古巴假霜霉菌孢子囊的计数。

Counting of cucumber downy mildew sporangia based on flow cytometry

In order to provide rapid counting method for pathogen epidemiology in time and space, and to establish disease prediction model, Pseudoperonospora cubensis, the pathogen of cucurbit downy mildew, was taken as an example of oomycetes to study the sporangia counting method using flow cytometry. The results showed that the combination of EDTA and SYBR Green I could label the sporangia with fluorescence. The optimum dyeing conditions of SYBR Green Ⅰ were studied. When the dyeing time was the same, the higher the concentration of fluorescent dye, the better the dyeing effect; when the concentration of fluorescent dye was the same, the staining ratio of sporangia increased with the extension of dyeing time. For the measurement of sporangium concentration in aqueous suspension, there was no significant differences between the result by flow cytometry and the one by microscope hemocytometer (P < 0.01). The correlation analysis showed that there was a linear correlation between the sporangium concentration measured by flow cytometry and the one by microscope hemocytometer (r = 0.993 4), suggesting that flow cytometry can be used to count sporangia of P. cubensis.