农林环境、食品药物等领域的许多科学问题都与细菌和真菌互作有非常密切的关系。为了揭示虫霉目真菌共生细菌多样性和生态功能，本项目选择蚜群中流行最广、最具代表性的虫霉目蚜虫专性病原真菌——新蚜虫疠霉为主要研究对象。首先通过利用16s rDNA-RFLP、FISH、显微注射技术、GFP标记等技术，结合科赫式法则，验证虫霉目真菌—细菌共生假设；其次采用PCR-DGGE，qPCR等技术方法，研究不同地理寄主来源、培养时间和抗生素处理等条件下的新蚜虫疠霉共生细菌的多样性、种群分布特征及其生物学效应。本研究对进一步揭示昆虫病原真菌与细菌的共生本质或内在的生态规律，拓宽真菌共生细菌的认识；阐明共生细菌与昆虫病原真菌的相关生物学功能的潜在联系；推动昆虫病原真菌资源或生态系统的开发利用，具有十分重要的意义。

Entomopathogenic fungi (epf) are unique in their ability to directly penetrate the insect cuticle and do not require ingestion, rendering them the only significant microbial pathogens of the piercing-sucking Homoptera, including aphids. The principal species of entomopathogenic fungi are found in the Ascomycota and Entomophthoromycota. When rated for virulence against aphids per se, the Ascomycota are considered ‘generalists’ and far inferior to the aphid-specialists in the Entomophthoromycota. However, as entomophthoroid-induced epizootics often develop among certain aphid species, and less so among others, it raises the question as to which factor/s govern fungal virulence? Preliminary research by the Chinese partner in this consortium has verified the existence of bacterial symbionts in the entomopathogenic fungus (epf), Pandora neoaphidis, a cosmopolitan species also reported from South Africa. The hypothesis is put forward that fungal virulence might be mediated by symbiotic bacteria; a notion supported by known attenuation of virulence following repeated in vitro subculture of these fungi. We will investigate the diversity of potential bacterial symbionts found inside fungal structures through 16s rRNA clone library construction, to illustrate the possible relationship between virulence of P. neoaphidis and diversity in the bacterial symbionts harboured within its hyphae and/or conidia. To make inferences about the vertical transmission of bacterial symbionts, we will compare the bacterial diversity in the hyphae versus primary conidia of P. neoaphidis. To understand the relationship between fungal virulence and bacterial symbiont diversity, we will compare the predictive 16s rRNA clone library and the virulence of two genetically comparable isolates with different symbiotic status (i.e., a fungal isolate with versus without symbiotic bacteria). Surveys will be conducted in both the northern (China) and southern (South Africa) hemispheres to collect isolates of entomophthoroid fungi actively parasitising aphids. The bacterial symbionts will be isolated and identified using 16s rDNA-RFLP as well as PCR-DGGE technology and their location verified through fluorescence in situ hybridization (FISH) (mainly Chinese partner) followed by LC50 and/or LT50 virulence comparison through laboratory bioassays (mainly South African partner). This study is novel, considering bacterial symbionts as potential virulence-mediating organisms within a tritrophic association. A better understanding thereof will enhance our attempts at employing these fungi for aphid biocontrol, either as bioinsecticides or in support of Conservation Biological Control.

农林环境、食品药物等领域的许多科学问题都与细菌和真菌互作有非常密切的关系。本项目通过中南（南、北半球）多株虫霉目菌株的采集、16s rRNA文库的构建和分析等研究揭示了虫霉目真菌共生细菌多样性；同时利用16s rDNA-RFLP、FISH、GFP标记、RNA-seq、激光共聚焦观察等技术，选择蚜群中流行最广、最具代表性的虫霉目蚜虫专性病原真菌——新蚜虫疠霉Pandora neoaphidis为主要研究对象，通过共生细菌与寄主真菌的科赫法则验证、共生细菌与真菌互作的转录组分析、以及具有较大多态性内生细菌的新蚜虫疠霉菌株F98028−与经抗生素处理后（多态性相对小）的新蚜虫疠霉菌株F98028+的毒力比较等研究，较完整证明了项目提出的虫霉目真菌—细菌共生假设：新蚜虫疠霉存在共生细菌并具有多样性，且共生细菌的存在和变化与新蚜虫疠霉的生物学效应（如寄主专化性、毒力增强/退化等）及生态功能具有密切的关系。本研究对进一步揭示昆虫病原真菌与细菌的共生本质或内在的生态规律，拓宽真菌共生细菌的认识；阐明共生细菌与昆虫病原真菌的相关生物学功能的潜在联系；推动昆虫病原真菌资源或生态系统的开发利用，具有十分重要的意义。