【作者】 彭国雄；

【导师】 夏玉先；

【作者基本信息】 重庆大学 ， 生物医学工程， 2008， 博士

【摘要】 我国是世界上蝗灾发生最为频繁的国家之一,其中东亚飞蝗是导致我国蝗灾发生的首要害虫。目前,我国蝗虫的主要控制手段是化学防治,化学农药大量长期施用已经造成蝗区严重的环境污染,使蝗区生态平衡受到破坏,蝗虫灾害的发生与治理进入恶性循环。使用与环境友好的生物农药替代化学农药成为今后控制蝗虫的重要发展方向。金龟子绿僵菌是一种昆虫病原真菌,具有安全、不杀伤非目标生物、害虫难以产生抗药性等优点,在蝗虫生物防治中起着重要作用。杀蝗绿僵菌生物农药作为国际蝗虫防治中最重要的可持续的生物防治手段,已被联合国粮农组织(FAO)推荐为环保产品推广应用。我国尚无登记注册的真菌杀虫剂农药。缺乏具有自主知识产权的杀蝗绿僵菌制剂是阻碍我国大规模应用绿僵菌制剂防治蝗虫的主要原因之一。真菌农药存在菌株选育困难、生产工艺研制滞后、缺乏质量控制体系等问题,这严重制约着杀虫真菌制剂的生产、研发和检测,目前国际上只有两个杀蝗绿僵菌生物农药登记注册。缺乏快速准确的毒力评价方法成为制剂研究的重要制约因素之一。昆虫病原真菌的入侵速度与在寄主体增殖是寄主昆虫罹病死亡的前提,其增殖数量及速率直接影响致病过程及杀虫效率。建立昆虫体内病原真菌的早期定量检测方法,能满足菌株选育、制剂研制以及制剂质量评价对杀虫剂毒力评价的需要,为我国杀虫真菌制剂的生产、研发和检测提供技术支持。本研究以分离于自然感染黄脊竹蝗Ceracris Kiangsu的金龟子绿僵菌CQMa102为实验菌株,主要内容包括:a)CQMa102的生物学特性研究;b)CQMa102的固体发酵参数优化;c)制剂技术研究与环境安全性评价;d)CQMa102杀蝗制剂田间应用技术研究;e)东亚飞蝗体内绿僵菌的荧光定量PCR检测体系的建立及应用。主要研究结果如下:1、通过与IMI330189非洲杀蝗菌株的生物活性、耐热性与抗紫外光线等生物学特性比较研究表明:CQMa102菌株对东亚飞蝗的生物活性高于IMI330189菌株,LT95提前1.3d;耐热性与抗紫外光线与对照菌株无显著差异。另外,CQMa102对蝗虫具有专化性。2、CQMa102菌株固体发酵优化培养基为:以大米为发酵物料,按1:0.3加入0.01-0.02%CuSO4、1%硝酸钠、1%微量元素混合液;培养条件为:发酵温度为25-28℃,初始pH值为5.5-6.5,发酵前期保持高湿、后期降湿,发酵周期12-15天。在优化培养基与培养条件下采用透气发酵袋发酵的产孢量比塑料发酵袋提高20%左右,平均达到19亿孢子/克。3、在选择色拉油为溶剂油的基础上添加3%的凡士林配制成稳定的100亿孢子/ml杀蝗绿僵菌油悬浮剂。常温贮存12个月孢子萌发率大于80%;能明显增加绿僵菌杀蝗生物活性。经原药与制剂的毒理学测评,绿僵菌孢子粉和100亿孢子/ml绿僵菌油悬浮剂属微毒类生物农药,对非目标生物友好。4、经过复配制剂兼容性筛选、增效实验确定含1%高效氯氰菊酯的100亿孢子/ml高氯?绿僵菌油悬浮剂,其共毒系数为263;喷施后第5天东亚飞蝗的死亡率达到90%以上,比100亿孢子/ml绿僵菌油悬浮剂处理组提前7-8d。与稀释液按1:4混合后施用,降低化学农药用量90%以上,减少对环境的非目标生物影响。5、真菌稀释液的最佳工艺参数:稀释液中混合乳化剂(HLB=4)、水与色拉油的重量百分比分别为1.5%、40%、58.5%。稀释液增效实验结果表明:稀释液缩短孢子萌发(90%以上)时间3小时以上;加快孢子侵入速度,点样接种后第4天,东亚飞蝗体液孢子平均浓度达2.41×107孢子/ml,为对照的约50倍;按2:1与杀蝗绿僵菌油悬浮剂混合后喷雾对东亚飞蝗的LT90提前1.44d。6、在多种生态环境中,100亿/ml杀蝗绿僵菌油悬浮剂与真菌农药稀释液按1:2混合后采用超低容量喷雾技术进行地面与飞机防治试验能有效控制3-4龄东亚飞蝗蝗蝻的种群数量;地面防治施药后9-15天防效达到90%以上,飞机防治施药后14-15d后残虫存活率<10%。7、根据绿僵菌28s rDNA的转录间隔区(ITS)和5.8S的基因序列,设计检测感病蝗虫体内绿僵菌菌体DNA分子的特异引物,通过优化感病蝗虫菌体DNA快速制备方法,建立了基于SYBR Green I的荧光定量PCR的定量检测体系。该检测方法专一、灵敏,检测下限为10 pg/μl绿僵菌,并且能从罹病36-48小时的东亚飞蝗血淋巴中早期定量检测到在虫体血淋巴中增殖的绿僵菌DNA。应用该技术研究稀释液增效作用进一步表明:稀释液的增效作用是通过加快绿僵菌穿透蝗虫表皮进入体内,使其在体内增值时间缩短实现的。更多还原

【Abstract】 East Asian locust, Locusta migratoria manilensis (Meyen), is listed as major pest of crops and pastures in China. Locust has been mainly controlled with large amount of broad-spectrum chemical pesticides that usually cause pollution of environment and damage to human beings, as well as result in an exacerbation of locust problems due to the loss of natural enemies. In order to reduce both locust damage and insecticide application, it is important to develop biological alternative agents as part of a program of integrated pest management (IPM) of locusts and grasshopper.Metarhizium anisopliae, an entomopathogenic fungus, play an important role in biological control of pests for its target-specific and pest resistance difficultly produced. Metarhizium anisopliae became one of the most promising biological agents in controlling locusts and grasshoppers and widely applied as safety commodity commended by Food and Agriculture Organization of the United Nations (FAO).However, Metarhizium anisopliae did not apply to control Locusts in large scale in China for lack of registered Metarhizium anisopliae agents and commercially marketed. There are some obstacles inhibited the application in large scale and development of the mycoinsecticide in mycoinsecticide, such as lag in research of process and lack of quality control system. One of the main reasons was the lack of methods to quick and accurate assessment of virulence. The gradually proliferation of entomopathogenic fungus in host is preliminary of pathopoiesis and the proliferation quantity and velocity affect directly to the pathogenic process and the pesticide efficiency. It will meet the needs for research and quality assessment of agents and virulence assessment during supervising to set up the methods of quantified determination to the early infecting stage of entomopathogenic fungi in insect. The methods will supply technologic support to the production, research and determination of mycoinsecticide.In this research, the entomopathogenic fungus strain CQMa102 distinguished as M. anisopliae var. acridum was isolated from cadivour of Ceracris Kiangsu (Orthoptera: Acrididae) in China. The research items were included a) Study on biological characters of strain CQMa102, b) Optimization the fermentation of strain CQMa102 industrialization on liquid-Solid phase, c) Establishment of formulation and assessment safety on environment, d) Application technology of strain CQMa102 in field, and d) Setup real-time fluorescent PCR system which detect strain CQMa102 in locusts. The main results achieved in the following aspects. 1. In the biological characteristic reseach of CQMa102,the insecticidal activity controlling L. migratoria manilensis (Meyen), heat-resistance of conidia and ultraviolet (UV)-resistance were compared with that of strain IMI330189 in laboratory. The results show there are no significant difference in heat-resistance and ultraviolet (UV)- resistance of conidia between two strains, but insecticidal activity controlling L. migratoria manilensis (Meyen) using strain CQMa102 is higher than that of strain IMI330189. Time of efficacy over 90% in the field cages test shortened 1-2 days. Otherwise, strain CQMa102 can infect several kinds of locusts and grasshoppers.2. The optimum culture medium, which was used in solid-state fermentation of strain CQMa102, was based on rice supplemented with 0.01-0.02% sporlulation agent CuSO4 1%NaNO3 and 1% liquid dissolving various trace elements. The optimum culture conditions of strain CQMa102 were among 25-28℃, initial pH5.5-6.5 of medium, higher ambient humidity in fermentation prophase and low humidity in culture period of 12-15 days. In optimum culture medium and condition, the yields reach 1.9×109 conidia/g of conidia of CQMa102 in ventilated ferment bag and increased about 20% compared with that in plastic bag. In addition, the illumination had no effect on sporulation of M. anisopliae strain CQMa102.3. The fine suspension plant oil-based formulation of Metarhizium anisopliae were formulated 1×1010 conidia/ml using soybean oil containing 3 % vaseline. The germination rate of conidia in formulation was 88% after 12 months storage at 26℃. The percentage of formulation suspending was 92.3% after 3 months storage at 26℃. The percent of mortality of locust reached 80% after treatment 5 days using formulated conidia of CQMa102 and was higher than that of unformulated conidia powder and liquid suspending conidia. The results of toxicity test of show that oil-based formulation of M. anisopliae belong to almost non-toxicity insecticide. The storage stability of conidia mixed with 5-10% soybean is significantly better than that of dry-conidia. The ultraviolet radiation resistance and heat-stability of oil-conidia mixed with diluentent of mycoinsecticide were higher that of dry-conidia and water-conidia.4、The 1% beta-cypermethrin exhibit strong synergism with 1×1010 conidia/ml M. anisopliae oil formulation. The syntoxicity coefficient of the combination is high up to 263. The efficacy of the combination reached >90% at 5 day after treatment against L. migratoria manilensis(Meyen) and shorten 7-8 days compared with M. anisopliae in oil-based formulation using spray. The dosage of beta-cypermethrin in the combination, which was mixed with diluentent of mycoinsecticide at 1:4 ratio before application, is lower than that of chemical insecticide of beta-cypermethrin. Accordingly beta-cypermethrin synergism with M. anisoplia reduced the effect on non-target biology in the field due to greatly reducing chemical application dosage.5. 4 Value of hydrophilic-lipophilic balance (HLB) of plant oil-water system was determined. The diluentent of mycoinsecticide, which hasten germination of conidia and enhance virulence of M. anisoplia against locusts, is consist in water (40%), soybean oil (58.5%) and composite emulsification (1.5%) chosen according to the principle of HLB. Results show that the concentration of spores of in locust is 2.41×107 spores /ml at 4 day after inoculating M. anisoplia mixing with the diluentent on locust, and approximate 50 times of that of M. anisoplia.6. In a wide variety of vegetation and weather condition, 2-4 instars nymph of Locusta migratoria manilensis (Meyen) were effectively controlled by strain CQMa102 of Metarhizium anisopliae formulated 1×1010 conidia/ml oil based formulation, which was mixed with diluentent of myco-insecticide with the ratio of 1:2, using a ULV. In ground spray trial against L. m. manilensis (Meyen), it took 9-15 days to caused 90% mortality. In the aerial spray against L. migratoria manilensis (Meyen), the final percent survivals of locusts lowered than 10% at 14-15 days. The strain CQMa102 of Metarhizium anisopliae formulated oil furmulation and powder effectively declined quantity of Ceracris Kiangsu (Orthoptera: Acrididae) in nature. The efficacy reached over 80% in 11 days after treatment.7. According to the internal transcription spacer(ITS) sequence of 23S rDNA and the gene sequence of 5.8S rDNA of Metarhizium anisopliae var. acridum CQMa102, a specific primer pair of fluorescence quantitative PCR was designed, and a modified extraction method of DNA from the infected haemolymph of male adult locust was optimized. By combination of the specific primer pairs and the DNA extraction method, a pre-mycoses quantitative detection approach for rDNA of insect pathogen in the haemolymph of infected locust was established using real-time fluorescent quantity PCR (FQ-PCR) which is of speediness, sensitiveness and specificity. With this method, rDNA of Metarhizium anisopliae in the haemolymph of locust could be detected 36 hrs post-inoculation.更多还原