【作者】 张涛；

【导师】 宁君；

【作者基本信息】 中国农业科学院 ， 农药学， 2011， 博士

【摘要】 绿盲蝽Apolygus lucorum是我国黄河流域和长江流域地区棉花产区的重要害虫,除危害棉花外,绿盲蝽在枣、葡萄、樱桃、桃、苹果、茶树、杨树苗和马铃薯等其它作物上危害也十分严重,一般年份能够损失可达到20-30%,严重年份超过50%。同时,绿盲蝽抗药性非常严重,危害严重地区每年需要喷洒化学农药10余次。大量使用农药,在杀死天敌的同时,给环境及人类健康造成了潜在危害。鉴于大面积爆发的绿盲蝽缺乏有效的防治方法,我们提出利用性信息素来作为监测和防治绿盲蝽的手段。本研究利用气相色谱(GC)、气相色谱-质谱联用(GC-MS)、触角电位仪(EAG)、气相色谱-触角电位联用(GC-EAD)和田间试验等方法与技术,首次对我国绿盲蝽的种内化学通讯进行了系统的研究,旨在探明我国绿盲蝽性信息素通讯及其动态变化规律,并为利用性信息素防治绿盲蝽提供理论支持和试验依据。主要结果如下:1.绿盲蝽性信息素的提取和鉴定通过气相色谱-质谱联用(GC-MS)分析,雌性绿盲蝽二氯甲烷提取物中的主要成分有三种:4-氧代-反-2-己烯醛、丁酸己酯和丁酸-反-2-己烯酯,其含量分别为21.75±2.04μg/头、1.42±0.56μg/头和23.74±1.89μg/头。而雄性绿盲蝽二氯甲烷提取物中主要成分与雌虫相同,但其比例有所不同,具体含量分别为13.68±3.32μg/头、27.73±6.54μg/头和3.84±1.67μg/头。丁酸己酯和丁酸-反-2-己烯在雌雄体内的比例完全相反,丁酸己酯为雄性绿盲蝽分泌的反性信息素。2.性信息素的释放规律绿盲蝽性信息素的释放量从暗周期开始到暗周期后2小时内没有明显变化;但从暗周期后2小时开始,提取物中4-氧代-反-2-己烯醛和反-2-丁酸己烯酯的含量迅速减少,至暗周期后约5小时其含量减少至最低点,说明绿盲蝽性信息素的释放高峰期可能在暗周期开始后2h-4h。光周期时雌性绿盲蝽体内性信息素含量逐渐增加,光照10h后即可达到4-氧代-反-2-己烯醛和反-2-丁酸己烯酯含量达到较高水平。说明绿盲蝽性信息素产生后并非立即释放出来,而是先在体内贮存,待暗周期求偶时释放。雌性绿盲蝽提取物中丁酸己酯的含量非常低,平均含量低于0.5μg,同时与暗周期、光周期没有明显的关系,没有明显规律性。3.性信息素的合成及活性测定利用化学合成受到对绿盲蝽提取三种主要组分:4-氧代-反-2-己烯醛、丁酸己酯和丁酸-反-2-己烯酯进行了人工合成,并通过核磁共振(HNMR)和质谱(MS)进行结构确认。通过触角电位(EAG)和气相色谱-触角电位联用(GC-EAD)对人工合成的性信息素进行了初步活性测定,证明三种人工合成化合物均能引起雄性绿盲蝽的触角反应,其中丁酸己酯的EAG反应最强,达到6.36±0.54mV,同时三种组分的二元、三元混合物的EAG活性显著高于单一组分。但是在Y形管嗅觉仪测定行为试验时,无论是单一组分还是二元、三元组分均不能明显吸引雄性绿盲蝽。4.田间诱捕试验及配方优化以实验室人工合成的性信息素化合物配制成自制诱芯进行田间诱捕试验,结果发现,单一组分诱捕到雄性绿盲蝽数量非常少,同时非常不稳定。而4-氧代-反-2-己烯醛和丁酸-反-2-己烯酯配比在一定范围时,能够诱捕到大量雄性绿盲蝽。通过田间试验对性诱剂配方进行优化,得出4-氧代-反-2-己烯醛和丁酸-反-2-己烯酯的比例在900:600时诱捕效果最好。丁酸己酯对诱捕效果没有贡献,并且会抑制对雄虫的吸引。5.反性信息素的研究丁酸己酯能够明显抑制性诱剂对雄性绿盲蝽的吸引效果,并且有非常高的活性。在由4-氧代-反-2-己烯醛和丁酸-反-2-己烯酯配组成的诱芯中,加入不同浓度的丁酸己酯,可以看出随着丁酸己酯量的增加,诱捕数量明显减少。当丁酸己酯达到性诱剂有效成分10%以上,性诱剂几乎失去活性。雌雄绿盲蝽交配后,雄性绿盲蝽将之注入雌虫体内或者诱导雌雄绿盲蝽产生丁酸己酯,其作用在于干扰其他雄性绿盲蝽对该雌虫的化学通讯定位,阻止其他绿盲蝽与该雌虫二次交配。6.其他盲蝽性信息素的初步研究采用同样的提取和鉴定方法,对中黑盲蝽Adelphocoris suturalis、三点盲蝽Adelphocoris fasciaticollis和苜蓿盲蝽Adelphocoris lineolatus的性信息素进行了初步鉴定,发现其主要组分同样为4-氧代-反-2-己烯醛、丁酸己酯和丁酸-反-2-己烯酯,但与绿盲蝽相比三种盲蝽中4-氧代-反-2-己烯醛含量很少。其中雌性中黑盲蝽和三点盲蝽中以丁酸己酯为主,雌性苜蓿盲蝽提取物中以丁酸-反-2-己烯酯为主。本研究首次鉴定和分析了绿盲蝽的性信息素的主要成分及其精确含量和比例,首次发现绿盲蝽中反性信息素的存在,并对其干扰绿盲蝽交配进行了验证试验。通过EAG反应、田间诱捕试验验证了性信息素的活性成分,获得了最佳诱芯的配方,可以直接应用于绿盲蝽的田间大量诱捕。本研究丰富了昆虫性信息素研究的内容,为绿盲蝽的高效、无毒、无污染、无公害防治提供了新方法、新途径。更多还原

【Abstract】 Apolygus lucorum is a significant kind of pest in the Huanghe Valley and Longriver Valley of China. Besides damaging cotton, the pest seriously damage the other crops such as jujube, grape, cherry, peach, apple, tea plant, poplar and potato. The loss can reach 20-30% of the above crop planted in the common years, and can exceed 50% in some years. Moreover, Apolygus lucorum are very restricted to drugs, so the disaster areas need to spread pesticide for more than 10 times, which killed the pests but at the same time cause potential damage to the environment and human health.Considering there were no effective ways to prevent the extensive outbreak of Apolygus lucorum, we came up with the idea to use sex pheromone to monitor and control Apolygus lucorum. In this research we applied the methods of GC, GC-MS, EAG, GC-EAG and field tests to find out how chemical communication mechanism of Apolygus lucorum works and explored their dynamic change regulation. All of these will offer theoretical support and experimental basis for the prevention and treatment to Apolygus lucorum. The main results are showed as follows:1, Extraction and identification of the sex pheromone from Apolygus lucorumAccording to GC-MS, the female sex pheromone from Apolygus lucorum mainly consists of 4-oxo-(E)-2-hexenal, hexylbutyrate, trans-2-hexenylbutyrate, their contents were 21.75±2.04μg, 1.42±0.56μg , 23.74±1.89μg respectively。The key components of male sex pheromone were the same compared with the female except their proportion, Which were 13.68±3.32μg、27.73±6.54μg and 3.84±1.67μg respectively. The ratio of hexylbutyrate and Trans-2-hexenylbutyrate were totally opposite between the male and female pest, so hexylbutyrate was anti-sex pheromone of the male.2, Releasing pattern of sex pheromoneThe amount of sex pheromone remained unchanging during the first two hours of the dark period; then 4-oxo-(E)-2-hexenaland, trans-2-hexenylbutyrate decreased rapidly until the fifth hour reach to the bottom amount. This phenomenon show that the releasing peaks of the pests’sex pheromone may probably be between the second to the forth hour .During the light period the sex pheromone in female pest gradually increased, until the tenth hour the amount 4-oxo-(E)-2-hexenal and trans-2-hexenyl butyrate reached to a very high level. This illustrated that Apolygus lucorum don’t release their sex pheromone immediately after producing .The pests were kept the sex pheromone in body and then released until mating call in dark period. The content of hexylbutyrate abstracts from female was really low, averagely lower than 0.5μg, which also had no obvious relationship with dark-light period.3. Synthesis and bio-activities of sex pheromoneWe synthesized the three main components abstracted from Apolygus lucorum: 4-oxo-(E)-2-hexenal、hexylbutyrate、and trans-2-hexenylbutyrate by using chemical methods, and verified their saturation through HNMR and MS. Then their activities were preliminary tested with EAG and GC-EAD. All synthetic compounds can cause antennal reaction and hexylbutyrate had the strongest EAG activity which reached 6.36±0.54mV. And the activity of the binary and the ternary components was better than the single component. However, when doing behavior test in Y-tube olfactometer, any of the components can’t obviously attract the male.4, Field test and the optimization of formulationThe synthetic sex pheromones were used to make lures in field test. The result showed that the single component was unstable and can only attract small number of the male Apolygus lucorum. One the other hand, the components of 4-oxo-(E)-2-hexenal and trans-2-hexenylbutyrate within a certain range can get good results. Through optimization test we noted that the best ratio of 4-oxo-(E)-2-hexenal and trans-2-hexenylbutyrate was 900:600. Hexylbutyrate had no contribution to attraction but had inhibitory action to the male pests.5, Research of anti-sex pheromoneHexylbutyrate can apparently restrain the sex pheromone’s attract to the male Apolygus lucorum and had strong activity. When it was added to the lures consisting of 4-oxo-(E)-2-hexenal and trans-2-hexenylbutyrate with different amounts, we could easily find that with increasing of hexylbutyrate, the number of attracted male pests decreased obviously. When the content of hexylbutyrate reaching to 10%, the sex pheromone almost turned inactive. After mating, the male bugs would inject hexylbutyrate into the female or tempted the female to produce hexylbutyrate, which would disturb other female’s orientation and prevent the second mating.6, Preliminary study of sex pheromone of other bugUsing the same extraction and identification method we study the sex pheromone of Adelphocoris suturalis, Adelphocoris fasciaticollis, and Adelphocoris lineolatus. Their main components were similarly 4-oxo-(E)-2-hexenal、hexylbutyrate and trans-2-hexenylbutyrate. However, compared with Apolygus lucorum, the content of 4-oxo-(E)-2-hexenal of the above three bugs was lower. In female Adelphocoris suturalis, the main component was Hexylbutyrate and the main components in female Adelphocoris lineolatus was trans-2-hexenylbutyrate.This research firstly verified and analyzed the main components of sex pheromone of Apolygus lucorum and their accurate ratio. At the same time we found out the presence of anti-sex pheromone, and verified its disturbance to the mating behavior. Through EAG reaction and field attract test we got the best formulation of tempts core, which can be directly used in the field attract. This research has enriched the knowledge of sex pheromone of insects, and will offer a new non-toxic controlling way of high efficiency, non-poisons, and non-environment pollutions.更多还原