橘小实蝇肠道引诱菌的筛选及其对宿主影响的研究

The oriental fruit fly, Bactrocera dorsalis (Hendel), is a quarantine pest worldwide of fruit plants, including citrus and other subtropical fruits. B. dorsalis is highly polyphagous, and can infest more than 250 species and varieties of host plants, including a number of commercially grown fruits, such as melon, banana, and mango. A growing body of research suggests that the gut microbes have been developed a close symbiosis relationship with B. dorsalis. It has been reported that isolated enteric bacteria from the alimentary tract of lab reared and wild B. dorsalis, and observed an olfactory attraction response to the bacterial isolates by B. dorsalis adults. In this project, bacterial community inhabiting the intestinal tract of adult oriental fruit flies (B. dorsalis) from Ganzhou, Jiangxi Province will be identified by the conventional method, API Kit, 16S rRNA and other bacterological technology and the attractive symbiotic bacteria (closely related sequences (>97% sequence similarity)) from Ganzhou, Wuhan, laboratory-reared and laboratory sterile sugar-reared populations will be found by homogeneous analysis. The attractive symbiotic bacteria and corresponding particular chemicals will be identified by attractancy bioassays in the laboratory and field tests, activated carbon solid phase microextraction (ACF-SPME) and gas chromatography-mass spectrometry (GC-MS). The interaction mechanism between the attractive symbiotic bacteria and B. dorsalis will be also studied with electroantennogram (EAG) and sterile insect culture techniques. Consequently, the expected results of this project may significantly contribute to the available information on bacterial isolates and the biological control of B. dorsalis.

橘小实蝇是一种世界性的检疫性害虫，能危害柑橘类、甜瓜、香蕉等250种水果和蔬菜。越来越多研究表明实蝇肠道细菌与宿主间的紧密关系，肠道细菌可作为成虫食物来源，其培养体对雄蝇和雌蝇均表现出了显著的引诱作用。本研究将以江西省赣州市柑橘种植区的橘小实蝇（GZ）成虫为实验材料，采用传统微生物培养技术、API酶谱系统、16S rRNA及其它细菌学方法分析其肠道可培养细菌群落结构，在97%序列相似性源上找到该种群与武汉种群（WH）、实验室种群（LR）和实验室无菌糖水喂养种群（LSSR）肠道共生细菌种类；通过共生细菌引诱实验、固相微萃取以及气相色谱-质谱联用（GC-MS）等方法，筛选引诱菌并分析引诱化学成分；通过触角电位反应（EAG）和无菌生物学技术，研究可诱捕菌群同实蝇间的相互作用机制。研究结果将为探索橘小实蝇与其肠道细菌的共生关系，阐述肠道细菌对宿主的功能，开发生物活性引诱剂等方面提供重要的科学依据。

橘小实蝇（Bactrocera dorsalis）是一种极具危害性的检疫性害虫，可危害柑橘类、甜瓜、香蕉等250种水果和蔬菜。通过研究肠道可培养细菌群落结构组成，肠道细菌对寄主成虫的引诱作用、存活率和寿命的影响，可为探索橘小实蝇与其肠道细菌的共生关系，阐述肠道细菌对宿主的功能，开发生物活性引诱剂等方面提供重要的科学依据。.本研究通过利用16S rRNA基因的PCR-DGGE分析技术研究了橘小实蝇四个种群成虫肠道肠道可培养细菌的群落结构组成；肠道细菌对寄主成虫的引诱作用；肠道细菌通过饲喂菌悬液的方式对寄主实蝇死亡率和寿命的影响。主要研究结果如下：.从橘小实蝇四个种群成虫肠道800株可培养细菌得到89种不同细菌遗传型，分属于肠杆菌科（Enterobacteriaceae）、肠球菌科（Enterococcaceae）、芽孢杆菌科（Bacillaceae）和链球菌科（Streptococcaceae）等四个科。其中肠杆菌科是肠道可培养细菌最优势的细菌种类。同样以序列相似性大于97%的菌株归为相同的细菌种类为标准，找到了除赣州种群外的其它三个种群可培养细菌的共有菌种，结合菌落形态观察和生理生化特征鉴定，确定共有菌种为肠杆菌属5株，克雷伯氏菌属2株，柠檬酸杆菌属1株，泛菌属1株，肠球菌属2株，以及芽孢杆菌属4株。.室内引诱实验发现，所有供试菌株的不同处理发酵液对橘小实蝇均有显著的引诱作用，其中以Bacillus cereus，Enterococcus faecalis，Enterobacter cloacae和Citrobacter freundii四种菌的引诱效果最好。野外引诱实验结果表明：六种菌的引诱效果都好于对照，其中B. cereus和C. freundii引诱到了最多的雌性、雄性和雌性加雄性实蝇，并且除Klebsiella oxytoca（引诱到的雌蝇和雄蝇一样多）外其余五种菌引诱到的雌蝇都多于雄蝇。引诱成分分析结果表明：分离到的成分吲哚、2,4-二(1,1-二甲基)-苯酚、N6-(4-甲氧基苯甲酰基)腺苷对橘小实蝇均具有引诱作用。.饲喂实验结果表明：实蝇寿命（无论雌雄）不受喂食抗生素的影响。实蝇对上述两类菌的摄取明显地影响自身的死亡率和寿命。总体而言，饲喂蜡样芽孢杆菌对寿命有不利影响，而饲喂肠杆菌科细菌则有利。