橡胶树白粉病菌在疏水介质表面的侵染特征和RNA提取

将白粉菌孢子接种到石蜡膜(parafilm)介质表面,观察不同温度条件下橡胶树白粉菌(Oidium heveae B.A.Steinmann)菌株孢子在疏水介质上的侵染过程,确定0~24 hpi孢子侵染结构发育特征,提取侵染发育过程白粉菌的总RNA,采用RT-PCK扩增分析18S保守基因并评价其质量。结果表明:橡胶树白粉菌孢子在15~34℃温度条件下接种于parafilm介质表面能萌发并形成附着胞,萌发和附着胞形成的适温范围22~28℃,最适离体诱导温度22℃。孢子在parafilm介质表面0~24 hpi(hour Post moculation)侵染发育过程可分为未萌发(0hpi)、萌发(4 hpi)、附着胞形成(6,8 hpi)和次生附着胞形成(16,24 hpi)等4个阶段;采用改良Trizol方法提取白粉菌孢子侵染发育过程4个阶段总RNA,可满足转录组文库要求,并扩增18S序列,进化分析显示其与白粉菌科物种为同一分支。结果表明,病原菌孢子在22~28℃温度条件下能够在介质表面形成侵染结构,0,4,6,8,16,24 hpi侵染发育阶段总RNA可用于转录组测序分析研究。

Total RNA Isolation and Observation of Infection Structures of Oidium heveae Induced by Hydrophobic Substratum

HO-73 strain of Oidium heveae B.A.Steinmann was cultured on parafilm surface at different hours post inoculation (hpi) under different temperatures to observe its infection process and the infection structure formation of its conidia.O.heveae was cultured at the temperatures ranging from 15 ℃ to 34 ℃ and the suitable temperatures for conidium germination and appressorium formation ranged from 22 ℃ to 28 ℃ with the optimal temperature being 22 ℃.The infection process of the conidia of the strain from 0-24 hpi was divided into 4 stages:no germination at 0 hpi,germination at 4 hpi,appressorium formation at 6,8 hpi and secondary appressorium formation at 16,24 hpi.Total RNA of the strain was isolated from the cultures at different infection stages from 0 hpi to 24 hpi.The 18Sr RNA was detected by RT-PCR at all the infection stages and analyzed by using the phylogenetic relationship.Total RNAs of the conidia at the four infection stages were isolated by using the modified Trizol method.The 18S rRNA of O.heveae was detected in all the conidia by RT-PCR,and clustered to the same branch as Erysiphaceae by using phylogenetic analysis.The result indicated that the infection structures of O.heveae were successfully developed on parafilm surface at a temperatures ranging from 22 ℃ to 28 ℃,and that the total RNA extracted from the cultures at 0,4,6,8,16 and 24 hpi can be used for 18S amplification.