【作者】 李斌；

【导师】 冯颖；

【作者基本信息】 中国林业科学研究院 ， 森林保护学， 2023， 博士

【摘要】 白蜡虫（Ericerus pela）是我国重要的资源昆虫,其二龄雄若虫分泌的白蜡具有重要的经济价值。当前,主要以女贞（Ligustrum lucidum）作为寄主植物用于白蜡虫的培育和白蜡的生产,“女贞-白蜡虫”经济林的栽培和养殖模式在区域生态文明建设和乡村振兴中的重要作用逐渐凸显。“昆虫-微生物-植物”多元体系互作的研究是当前昆虫学和植物学研究的热点和前沿。研究“白蜡虫-微生物-女贞”多元体系的互作关系,挖掘潜在的益生菌资源和识别有害微生物,可为白蜡虫的高效培育和“女贞-白蜡虫”经济林的可持续经营提供科学依据。本研究采用荧光原位杂交、PCR检测、扩增子和宏基因组测序等技术,探究了立克次氏体（Rickettsia）在白蜡虫中的分布模式和水平传播途径;分析了立克次氏体和其他微生物在白蜡虫生长发育和生态适应中的作用和功能;解析了女贞不同器官和根际微生物群落的组成和功能特征;探讨了驱动女贞内生菌群结构变化的生理因素和生态因子;初步揭示了“白蜡虫-微生物-女贞”三元互作关系。主要研究结果如下:（1）立克次氏体在白蜡虫体内呈“聚集型”和“分散型”分布,在调控白蜡虫的解毒、防御、营养代谢和生长发育中发挥着重要作用。立克次氏体主要分布于白蜡虫的含菌细胞、血淋巴、脂肪体及消化道中,其可在白蜡虫、白蜡虫花翅跳小蜂和长叶岱蝽间水平传播。宏基因组测序分析表明,立克次氏体可通过参与辅酶因子、维生素、能量、碳、氮和脂类代谢调控白蜡虫的营养代谢;通过合成糖基转移酶调控白蜡虫的解毒、适合度和生长发育;通过其分泌系统和产生粘附、入侵、抗吞噬作用等毒力因子在白蜡虫体内进行物质交换、定殖、扩散和繁殖;此外,立克次氏体还可通过种间竞争、分泌毒素和合成次生代谢产物来保护白蜡虫抵御天敌和病原微生物的危害。（2）四环素处理可有效清除白蜡虫体内的立克次氏体,进而改变白蜡虫内生菌群多样性、结构和功能特征,阻碍白蜡虫正常的生长发育、繁殖和存活。经1000 mg·L^-1四环素溶液处理后,白蜡虫的重量、怀卵量和存活率显著降低,泌蜡量无明显变化,内生细菌和真菌多样性降低,菌群结构和功能发生明显改变。立克次氏体被清除后,杀雄菌属和炭疽菌属在白蜡虫体内显著富集。除立克次氏体外,白蜡虫体内的甲基杆菌属、马赛菌属、线虫草属和枝孢属也对其生长发育发挥有益作用。（3）女贞叶、茎、根和根际土壤的细菌和真菌多样性、群落结构和功能特征具有相似性和差异性。根际土壤的细菌和真菌多样性显著高于根、茎和叶,叶与茎具有相似的内生细菌和真菌群落结构。在门级水平,变形菌门、放线菌门和子囊菌门为女贞根、茎和叶中的优势类群,而绿弯菌门和担子菌门在根际土壤中占优势。在属级水平,红球菌属、雷尔氏菌属、枝孢属在叶和茎内占优势,慢生根瘤菌属、伯克氏菌属、Rhexodenticula和普可尼亚属在根内占优势,热酸菌属和裸盖菇属为根际土壤的优势类群。叶、茎、根及根际土壤细菌群落的功能以化能异养为主,叶、茎菌群主要在碳、氢循环中发挥作用,而根和根际土壤菌群主要行使与氮循环相关的功能。在真菌功能群中,叶和茎中病理-腐生-共生营养型、病理-腐生营养型真菌相对丰度较高,而根和根际土壤中腐生营养型、共生营养型真菌占比较高。（4）白蜡虫取食胁迫诱导女贞生理代谢响应,进而驱动女贞内生菌多样性、群落结构和功能发生变化。经白蜡虫取食后,女贞叶和茎中的茉莉酸（JA）、氨基酸（AA）、丙二醛（MDA）、植物蛋白酶抑制剂（PI）含量及内生真菌多样性显著升高。经取食90 d和150 d后,女贞茎内细菌和真菌群落结构、物种组成和功能特征发生显著改变,茎内变形菌门、子囊菌门的相对丰度显著降低,而放线菌门、担子菌门的丰度显著升高;茎内细菌群落在涉及碳和氮循环方面的功能丰度显著降低,病原真菌的丰度显著升高。在检测的12个植物生理指标中,JA、细胞分裂素（CTK）、脯氨酸（Pro）、MDA、过氧化物酶（POD）和可溶性糖（PSS）与女贞内生细菌和真菌群落结构存在显著相关性。Pro、PSS和MDA是影响女贞内生细菌群落结构变化的主要生理特性,而CTK、PSS和植物总酚（TP）是驱动女贞内生真菌群落结构分异的主要生理因子。综上所述,“白蜡虫-微生物-女贞”多重营养关系具有复杂性和多样性:白蜡虫优势共生菌立克次氏体可协助解毒寄主植物女贞合成的有毒化合物,提高白蜡虫的适合度;白蜡虫取食危害削弱了女贞树势,进而促进植物病原真菌（横断孢属）的定殖;女贞内生白蜡虫病原真菌可感染并杀死白蜡虫,降低白蜡虫的存活率;白蜡虫取食诱导女贞生理代谢响应,进而驱动女贞内生菌群结构的变化。此外,存在多种类别的细菌和真菌可能在土壤-女贞-白蜡虫间传递,并对女贞和白蜡虫产生有益或有害的影响,这些微生物介导女贞-白蜡虫间相互作用的分子机制还有待进一步研究。更多还原

【Abstract】 The white wax scale insect（Ericerus pela）is an important resource insect in China.The white wax secreted by the second-instar male larva has considerable economic value.At present,privet（Ligustrum lucidum）is the main host plant used for the cultivation of E.pela and the production of white wax.The economic forestry model of“L.lucidum–E.pela”is gradually emerging as an important role in the in the construction of regional ecological civilization and rural revitalization.The study of the interaction of multiple systems of insect-microbial-plant is a hot topic and frontier in entomology and botany.The research on the interactions between the binary and ternary systems of“E.pela-microorganisms-L.lucidum”,the exploration of potential probiotic resources and the identification of harmful microorganisms can provide a scientific basis for the efficient cultivation of E.pela and the sustainable management of“L.lucidum–E.pela”economic forest.In this study,we investigated the distribution pattern and horizontal transmission pathways of Rickettsia in E.pela,analyzed the main functions of Rickettsia and other microorganisms in the development and ecological adaptation of E.pela,analyzed the composition and functional characteristics of microbial communities in different organs and rhizospheric soil of L.lucidum,explored the physiological and ecological factors driving the structure change of endophytic microbial community in L.lucidum,and preliminarily revealed the“E.pela-microorganisms-L.lucidum”triadic interactions.The main results are as follows:（1）The distribution of Rickettsia in the body of the E.pela is characterized by both“aggregation”and“dispersion”,and serves a crucial function in the regulation of detoxification,defense,nutrient metabolism,as well as the growth and development of the E.pela.Rickettsia were mainly found in the bacteriocytes,hemolymph,fat body and digestive tract of E.pela,and could be horizontally transmitted among E.pela,Microterys ericeri and Dalpada jugatoria.Metagenomic sequencing analysis showed that Rickettsia can regulate the nutritional metabolism of E.pela by participating in the metabolism of coenzyme factors,vitamins,energy,carbon,nitrogen and lipids;can regulate detoxification,fitness and growth of E.pela by synthesizing glycosyl transferase;can carry out substance exchange,colonization,diffusion and reproduction in E.pela through its secretion system and the production of adhesion,invasion,anti-phagocytosis and other virulence factors.In addition,Rickettsia can protect the E.pela from natural enemies and pathogenic microorganisms by interspecific competition,secreting toxins and synthesizing secondary metabolites.（2）Tetracycline treatment could effectively eliminate Rickettsia,change the diversity,structure and function characteristics of endophytic microorganism,and hinder the growth,development,reproduction and survival of E.pela.After treatment with 1000 mg·L^-1 tetracycline solution,the weight,ovulation quantity and survival rate of E.pela were significantly decreased,the wax secretion was not significantly changed,and the diversity of endophytic bacteria and fungi was decreased.At the same time,the structure and function of endophytic microbial community were significantly changed.After Rickettsia was removed,Arsenophonus and Colletotrichum were significantly enriched in E.pela.In addition to Rickettsia,Methylobacterium,Massilia,Ophiocordyceps and Cladosporium also play a beneficial role in the growth and development of E.pela.（3）There were similarities and differences in the diversity,structure and function of bacterial and fungal communities in leaf,stem,root and rhizosphere soil of L.lucidum.The diversity of bacterial and fungal communities in rhizosphere soil was significantly higher than that in root,stem and leave.The leave and stem had similar endophytic bacterial and fungal community structure.At the phylum level,Proteobacteria,Actinobacteriota and Ascomycota were the dominant groups in root,stem and leave,while Chloroflexi and Basidiomycota were dominant in rhizosphere soil.At the genus level,Rhodococcus,Ralstonia and Cladosporium were dominant in leave and stem,Bradyrhizobium,Burkholderia,Rhexodenticula and Pochonia were dominant in root,Acidothermus and Psilocybe were dominant in rhizosphere soil.The functions of leaf,stem,root and rhizosphere soil bacterial community were mainly chemoheterotrophic.The microorganism in leaf and stem mainly played roles in carbon and hydrogen cycle,while microorganism in root and rhizosphere soil mainly played roles in nitrogen cycle.Among the functional groups of fungi,the relative abundance of pathotrophy-saprotrophy-symbiotrophy and pathotrophy-saprotrophy fungi was higher in leaves and stems,while saprotrophy and symbiotrophy fungi was higher in roots and rhizosphere soil.（4）The physiological and metabolic response of L.lucidum was induced by E.pela feeding.The diversity,community structure and function of endophytic microbes were changed.The contents of jasmonic acid（JA）,amino acid（AA）,malondialdehyde（MDA）and plant protease inhibitor（PI）in the leaves and stems of L.lucidum and the diversity of endophytic fungi increased significantly after feeding by E.pela.After 90 and 150 days of E.pela parasitization,the structure,species composition and functional characteristics of bacterial and fungal communities in the stems of privet changed significantly.The relative abundance of Proteobacteria and Ascomycota significantly decreased,while that of Actinobacteria and Basidiomycota significantly increased.The functional abundance of bacterial communities involved in carbon and nitrogen cycling was significantly decreased,while the abundance of pathogenic fungi was significantly increased.Among the 12 physiological indexes,JA,cytokinin（CTK）,proline（Pro）,MDA,peroxidase（POD）and plant soluble sugar（PSS）were significantly correlated with the community structure of endophytic bacteria and fungi in L.lucidum.Pro,PSS and MDA were the main physiological characteristics affecting the community structure of endophytic bacterial community,while CTK,PSS and total phenol（TP）were the main physiological factors driving the community structure differentiation of endophytic fungal community in L.lucidum.The multitrophic relationship between E.pela,microorganisms,and L.lucidum is both complex and diverse.The symbiotic bacterium Rickettsia,which is dominant in E.pela,may aid in the detoxification of toxic compounds synthesized by the host plant L.lucidum and improve the fitness of E.pela.However,the feeding behavior of E.pela can also weaken the tree growth potential of L.lucidum and promote the colonization of plant pathogenic fungi,such as those found in Strelitziana.The endophytic entomopathogenic fungi found in L.lucidum have the ability to infect and kill E.pela,which ultimately results in a reduced survival rate for the latter.Additionally,the feeding of E.pela triggers a physiological and metabolic response in L.lucidum,leading to alterations in the diversity,community structure,and function of endophytic microbes.In addition,there are various types of bacteria and fungi that may be transmitted between soil,E.pela and privet,which can have either positive or negative impacts on both species.However,the exact molecular mechanisms through which these microorganisms mediate the interaction between privet and E.pela require further investigation.更多还原