虫霉目蚜科专化菌在竹蚜种群内流行机制及其生防潜能探究

Fungi in the order of Entomophthorales are natural regulators for controlling pest populations in agriculture and forestry, which are important to maintain forest health. Researches on entomophthoralean application have no breakthrough due to little understanding about the potential mechanisms of their epizootics in fields. This research plans to adopt the methods of air captures of migratory alates and environmentally monitoring green fluorescent protein (GFP) tagged strain to explore the abovementioned problems. Based on the collected data on air captures and individual rear, the study aims to analyze the potential of pathogen-infected host alates in colonization and mycosis transmission and the possibility of initial inoculum resource. The isolated strain was transformed with recombinant plasmid which carries the gene for EGFP (an enhanced green fluorescent protein) as a molecular marker, in order to obtain a genetically stable GFP-tagged strain. Successively, bamboo aphids inoculated by the recombinant strain with the method of conidial shower would be released in sampling fields of aphid-infested bamboo forest, for screening the development of the fungus within its host aphid populations. Real time monitoring for the dynamics of the fungal temporal-spatial distribution in the aphid populations and this habitat is to be conducted over one year. This investigation aims to analyzing the interaction between the entomophthoralean fungus and the host populations and disclosing biotic and abiotic regulation on its epizootic mechanisms and the biocontrol potential of Entomophthorales against bamboo aphids. This study will make progress on applying entomophthorealen fungi to control pest in agriculture and forestry, and also provide new sight in the development of molecular ecological control in the field of forest protection.

虫霉目真菌是一类调控农林害虫种群的天然因子，在维持森林健康方面具有重要作用。由于对其野外流行机制研究尚浅，虫霉生物防治应用研究开展并不顺利。本研究计划采用空中诱蚜和绿色荧光蛋白GFP标记环境示踪法探究相关问题。通过诱蚜和单头饲养实验了解有翅竹蚜定殖能力、带菌情况和真菌病传播能力等，分析虫霉随寄主迁入传病作为起始侵染体来源的可能。同时，将携带gfp报告基因的重组质粒转化入所分离的虫霉菌株，获得稳定遗传表达的标记菌株。随后，将标记菌株通过"孢子浴"法接种竹蚜，释放入蚜害爆发的竹林中，使其定殖于样地蚜群中。开展为期一年的虫霉在竹林蚜群和生境中的时空分布的实时监测调查，用于分析虫霉与寄主种群以及各项环境因子间的相互作用关系，并探究潜在的流行机制和生防能力。这项研究的开展将促进利用虫霉目真菌资源防控农林害虫，也为在森林保护领域发展分子生态调控提供新的思路。

竹蚜是影响竹林健康和危害竹产业的主要害虫，多数种类具有繁殖快、迁飞等特性。虫霉目真菌在自然环境中通过真菌病在寄主种群内流行而控制种群密度，具有生防应用价值。本项目针对开发虫霉目蚜科专化菌对寄主竹蚜的防控潜能展开研究。通过调查迁飞竹蚜的携播天敌情况，验证了虫霉目真菌随寄主蚜虫迁飞传病的假说在林木系统内也同样成立。同时通过2014年的空中诱捕迁飞竹蚜和2015-2016年的野外观察实验，摸清浙江省内竹林分布区主要竹叶梢部竹蚜种类及其种群增长规律。在诱捕的3577头竹蚜中发现虫霉新接霉属真菌一新种，通过形态学和分子生物学鉴定，定名为临安新接霉；通过进一步室内饲养观察该菌在迁飞寄主中发生率远高于其它天敌包括2种寄生蜂和1种外寄生螨，且致死快。并在多地竹蚜种群中发现该菌，通过长期跟踪定点调查记录，初步得出临安新接霉真菌病在竹蚜种群的流行扩散规律，即“迁飞传病”和“原位传病”兼有。由于虫霉目蚜科专化菌在新接霉属的种类都无法体外培养，不能进行遗传操作，且未发现可用的遗传筛选标记，因此，gfp报告基因转化的实验研究进展有限。生物测定实验结果显示可培养的蚜科专化菌暗孢耳霉在四种主要竹蚜的致死率和抑制寄主生殖力上效果理想。该菌具有控制寄主种群增长的能力，但控制作用较为短暂，需间隔释菌保持控蚜作用，主因是该菌在寄主体内易形成休眠孢子。此外，利用现代转录组学分析了暗孢耳霉致病的关键基因及其表达情况，发现类枯草杆菌素和脘蛋白类蛋白酶基因多样性对该菌侵染寄主范围具有影响，是其致病的关键基因之一。今后将进一步在分子水平上探索该菌与寄主互作机制。项目执行3年期间，项目主持人以第一作者/通讯作者共发表科研论文6篇，其中SCI论文3篇，国内一级期刊论文3篇，超额完成预期指标。