基于苜蓿花叶病毒和白三叶草花叶病毒复合侵染苜蓿的协生作用及其机理研究

Mosaic virus disease is one of the main diseases that has been widely occurred in alfalfa planting area of Gansu, and the disease incidence was more than 80%. Moreover, alfalfa mosaic virus (AMV) is one of the main pathogens that infect the host of alfalfa. In our previous studies, we found that AMV and white clover mosaic virus (WCMV) could be co-infected alfalfa and aggravated the occurrence of virus diseases, which seriously affects the yield and quality of alfalfa. In the present research, the pathogens of AMV and WCMV will be selected as the research subjects in alfalfa growing areas, Gansu Province, the virus gene cloning and transformation, high-throughput sequencing and bioinformatics analysis, and the synergistic effects of AMV and WCMV will be studied from the aspects of disease symptoms, virus content and histopathological changes of host cells. Nicotiana benthamiana will be selected as a model plant to study the interaction mechanism between AMV and WCMV with the hosts, and also comparatively analyzes its genes differentially expressed during the co-infection with AMV and WCMV，and excavates the key genes and verifies its function，discovers the reasons for leading to the host symptoms aggravate with synergism, the metabolic pathways that affect the co-infection of N. benthamiana and analyze the possible mechanism of the synergism between AMV and WCMV by RNA-Seq and VIGS technology. Our results will provide the great significance for guiding the prevention and control of alfalfa virus disease, decreasing the harm and improving the yield and quality of alfalfa, and also lays a foundation for the research of alfalfa virus disease.

花叶病是甘肃省苜蓿上发生的主要病害之一，发病率高达80%以上，苜蓿花叶病毒（AMV）是主要病原，我们在前期工作中发现AMV和白三叶草花叶病毒（WCMV）可复合侵染苜蓿，加重病毒病害发生，严重影响苜蓿的产量和品质。本项目以复合侵染甘肃省苜蓿的AMV和WCMV作为研究对象，借助植物病毒克隆转化、高通量测序及生物信息学分析等技术，从病害症状、病毒含量及寄主细胞组织病理变化等方面研究这两种病毒的协生作用；以本氏烟作为模式植物，研究二者在寄主体内的互作机理；通过RNA-Seq和VIGS技术比较分析这两种病毒复合侵染本氏烟过程中差异表达的寄主基因，挖掘受协生作用影响的关键基因并验证其功能 ，寻找协生作用导致寄主症状加重的原因；分析两种病毒复合侵染影响本氏烟的代谢通路，解析其协生作用的可能机理。研究结果对指导苜蓿病毒病防治，减轻危害，提高苜蓿草的产量和品质，具有重要意义；同时对苜蓿病毒病的研究奠定基础。

花叶病是甘肃省苜蓿上发生的主要病害之一，发病率高达80%以上， AMV和白三叶草花叶病毒（WCMV）可复合侵染苜蓿，加重病毒病害发生，严重影响苜蓿的产量和品质。本项目以复合侵染甘肃省苜蓿的AMV和WCMV作为研究对象，借助植物病毒克隆转化、高通量测序及生物信息学分析等技术，从病害症状、病毒含量及寄主细胞组织病理变化等方面研究这两种病毒的协生作用。结果表明田间生长期苜蓿和实验室种植的苜蓿幼苗(苗龄30 d)均带有AMV和WCMV，且两种病毒的复合侵染率分别为80.0%和25.0%；确定了AMV、WCMV单独侵染和复合侵染最佳接种浓度分别为1∶4、1∶1和3∶1(AMV∶WCMV)，温度25℃、RH 60% 有利于复合侵染发病，症状表现为褪绿、斑驳、黄化等(21d)，叶绿素含量显著降低，且两种病毒相对表达量比其单独侵染提高了39.46%和31.53%；电镜观察发现存在杆菌状的AMV（20～75nm）和线状WCMV（400～600nm）两种病毒粒子，引起叶绿体破坏等严重的组织超微病变；AMV和WCMV复合侵染可显著提高本氏烟叶片中的POD、PPO、PAL、CAT防御酶活性和胞壁降解酶纤维素酶等的活性，促进叶片细胞壁胼胝质积累和气孔数量的增加；影响本氏烟光合气体交换参数及光合荧光特性和内源激素含量，可显著降低本氏烟叶片净光合速率、水分利用效率、Fm、Fv/Fm、Y(Ⅱ)、qP（13.23%～74.55%）和内源激素IAA、GA、ZR、JA的浓度（59.09%～64.56%）、提高叶片蒸腾速率、气孔导度、胞间CO2浓度、F0、qN（10.14%～40.27%）和ABA、SA、SLs、BR 、ETH的浓度（36.27% ～287.29%）；通过观察摩擦接种AMV的转WCMV-CP基因本氏烟发现发病症状与复合侵染一致。转录组测序证明两种病毒复合侵染可严重影响叶绿素、糖代谢等合成代谢途径，使与光合作用有关的53个基因发生差异表达，其中31个上调表达、22个下调表达，探明了AMV和WCMV复合侵染协生作用致病机理。同时获得了防效在60%以上的AMV弱毒株AMVa3、AMV和WCMV混合液的弱毒株AW23、AW38和宁南霉素、氨基寡糖素，明确了弱毒株与药剂的防病机理。研究结果对指导苜蓿病毒病防治，减轻危害，提高苜蓿草的产量和品质，具有重要意义；同时对苜蓿病毒病的研究奠定基础。