Wang Hongsu, Chen Qi, Liu Luqin, Zhou Yan, Wang Huanhuan, Li Zhongan, Liu Jinxiang
Citrus Research Institute, Southwest University, Chongqing 400712, China.
Sichuan Provincial Key Laboratory of Quality and Innovation Research of Chinese Materia Medica, Sichuan Academy of Chinese Medicine Sciences, Chengdu 610041, China.
Plant Pathol J. 2022 Aug;38(4):287-295. doi: 10.5423/PPJ.OA.01.2022.0011. Epub 2022 Aug 1.
Citrus tristeza virus (CTV) is efficiently transmitted in a semi-persistent manner by the brown citrus aphid (Toxoptera citricida (Kirkaldy)). Currently, the most sensitive method for detecting plant viruses in insect vectors is reverse-transcription quantitative polymerase chain reaction (RT-qPCR). In this study, the elongation factor-1 alpha (EF-1α) gene and acidic p0 ribosomal protein (RPAP0) gene were confirmed to be suitable reference genes for RT-qPCR normalization in viruliferous T. citricida aphids using the geNorm, NormFinder, and BestKeeper tools. Then the relative CTV titer in aphids (T. citricida) at different post-acquisition feeding times on healthy plants was quantified by RT-qPCR using EF-1α and RPAP0 as reference genes. The relative CTV titer retained in the aphids gradually decreased with increasing feeding time. During the first 0.5 h of feeding time on healthy plants, the remaining CTV titer in aphids showed about 80% rapid loss for the highly transmissible isolate CT11A and 40% loss for the poorly transmissible isolate CTLJ. The relative CTV titer in aphids during more than 12 h post-acquisition times for CT11A was significantly lower than at the other feeding times, which is similar to the trend found for CTLJ. To our knowledge, this is the first report about the relative titer variation of CTV remaining in T. citricida at different post-acquisition feeding times on healthy plants.
柑橘衰退病毒（CTV）可通过褐橘蚜（Toxoptera citricida (Kirkaldy)）以半持久性方式高效传播。目前，检测昆虫载体中植物病毒最灵敏的方法是逆转录定量聚合酶链反应（RT-qPCR）。在本研究中，使用geNorm、NormFinder和BestKeeper工具确认了伸长因子-1α（EF-1α）基因和酸性p0核糖体蛋白（RPAP0）基因是用于带毒褐橘蚜RT-qPCR标准化的合适内参基因。然后，以EF-1α和RPAP0作为内参基因，通过RT-qPCR对褐橘蚜在健康植株上不同获毒取食时间后的相对CTV滴度进行了定量分析。随着取食时间增加，蚜虫体内保留的相对CTV滴度逐渐降低。在健康植株上取食的最初0.5小时内，对于高传播性分离株CT11A，蚜虫体内剩余的CTV滴度迅速损失约80%，而对于低传播性分离株CTLJ则损失40%。对于CT11A，在获毒后超过12小时的时间里，蚜虫体内的相对CTV滴度显著低于其他取食时间，这与CTLJ的趋势相似。据我们所知，这是关于褐橘蚜在健康植株上不同获毒取食时间后体内剩余CTV相对滴度变化的首次报道。
