田间褐飞虱种群的细菌型共生菌多样性及其对致害性的影响

Rice is a primary food crop for people worldwide. It is vital to control the various insect pests that damage rice. The brown planthopper (BPH) is a monophagous insect herbivore of rice that feeds on rice phloem and causes serious damage to rice crops. BPH also transmits rice viruses, which are serious diseases in the tropical region. In recent years, BPH infestations have intensified in China, causing heavy rice yield losses. As the popular rice varieties are susceptible to BPH, farmers depend on chemical insecticides for controlling this insect, but the long-term application of insecticides may result in pest resurgence. Another strategy for BPH control is to use host-plant resistance by breeding rice varieties with BPH resistance genes. However, BPH has repeatedly shown the ability to rapidly adapt to resistant rice varieties after several generations of continuous rearing in the field and laboratory. It has been well recognized that diversity exists within insect species and that "biotypes" or populations that are adapted to or that prefer a particular host can frequently develop. In BPH, the biotype is assigned to a population with the ability of virulence to damage varieties of rice that carry resistance genes. However, the precise nature of the virulence-conferring mechanisms in BPH biotypes is not clear..Close associations between insects and the symbiont microbes they harbor appear to be common. Symbionts have been found to contribute to the nutrition, development, reproduction, speciation, and defense against natural enemies of their host insects. Like other phloem-feeders, BPH harbors symbionts, including eukaryotic and prokaryotic microbes. BPH reportedly harbors the intracellular, eukaryotic "yeast-like symbiote" (YLS) in the fat body, which is essential for survival and development of BPH. In our previous research, we examined the bacterial symbiont microbes in BPH and identified a total of 18 operational taxonomic units (OTUs), representing four phyla (Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes) from three biotypes populations of BPH (biotype I, II, III). In addition, the species and numbers of the detected OTUs differed substantially among the BPH populations..The major objective of this study is to investigate diversity of bacterial symbiont microbes of field population of BPH and whether the interactions between bacterial symbiont microbes and virulence exist in BPH populations or not. We will identify the differences of diversity of bacterial symbiont microbes of field population and laboratory population of BPH by sequencing and analyzing 16S rDNA gene libraries and BOX-PCR methods; the differences of virulence to rice between field population and laboratory population of BPH by analyzing honeydew excretions; the differences of expression patterns of rice defense-response genes to BPH after removing bacterial symbiont microbes from field population and laboratory population of BPH.

褐飞虱以刺吸式口器取食水稻韧皮部汁液，影响水稻生长发育和产量，褐飞虱还传播多种水稻病毒病，是我国水稻生产中的首要害虫。目前褐飞虱的多种生物型给防治带来了很大困难。昆虫与其体内的共生菌经过长期协同进化形成了互利共生的关系，共生菌对昆虫宿主的营养、发育、生殖、防御天敌和寄主选择等都起到重要作用。申请者前期对实验室褐飞虱种群的细菌型共生菌进行了鉴定研究，发现褐飞虱生物型和其细菌型共生菌间有明显的对应关系。本研究拟对贵州田间褐飞虱种群中细菌型共生菌的多样性进行研究，就其种类、体内分布情况和形态特征与实验室种群的进行比较，并分析褐飞虱田间种群和实验室种群中共生菌与对水稻致害性的关系，在此基础上进一步通过对褐飞虱作除菌处理，确认共生菌是否进入水稻体内并激发水稻防御反应。研究结果将有助于深入认识"水稻-褐飞虱-共生菌"互作关系，解明细菌型共生菌的生物学功能，为通过共生菌调控褐飞虱种群提供依据。

项目围绕褐飞虱的细菌型共生菌在“水稻-飞虱”的互作关系中的作用进行了研究。主要获得了以下结果：（1）对田间褐飞虱和白背飞虱的细菌型共生菌进行了16S rDNA文库建库和检测，在褐飞虱中发现12种共生菌，在白背飞虱中发现5种共生菌；（2）使用BOX-PCR的扩增技术扩增田间褐飞虱与白背飞虱，发现两种飞虱扩增带型差异较大，与16S rDNA文库结果有一定关联性；（3）对褐飞虱体内共生菌和褐飞虱取食分泌进入水稻叶鞘细菌进行了培养，褐飞虱体内共生菌的培养检测到3种细菌，分别为：Staphylococcus sciuri、Microbacterium kitamiense、Herbaspirillum seropedicae；褐飞虱取食分泌进入水稻叶鞘细菌的培养结果，对照组没有细菌克隆生长；处理组检测到2个泛菌属细菌Pantoea ananatis和Pantoea agglomerans，其16S rDNA序列相似性为99%；（4）使用高通量测序技术对抗生素处理前后褐飞虱转录组，以及抗生素处理前后褐飞虱取食的TN1水稻转录组进行了测序，对不同转录组的差异表达基因进行了研究。抗生素利福平处理后的褐飞虱转录组差异显示，大部分差异表达基因（占总数的97%）显示为上调，仅3%的差异表达基因表现为下调。正常褐飞虱与使用抗生素利福平处理的褐飞虱取食的水稻转录组测序分析表明，差异表达基因较少，仅有783个，其中表达上调的为316个，表达下调的为467个，根据生物信息学分析结果挑选了与水稻-微生物、水稻防御反应、信号转导途径的候选基因，正在进行验证分析；（5）此外，还使用高通量测序技术对抗生素处理前后褐飞虱体内细菌型共生菌的丰度和多样性进行了测序研究。通过对不同抗性品种（TN1、Mudgo和ASD7）水稻上的褐飞虱群体和不同抗生素（氨苄青霉素、卡那霉素和利福平）处理的褐飞虱群体进行数据分析，共分类到152个细菌的操作分类单元（OTUs）。在不同抗性水稻品种的褐飞虱群体中，细菌型共生菌种类和丰度差异较大；使用不同抗生素处理后，发现褐飞虱体内的共生菌种类和丰度也发生了较大变化。