杀蝗绿僵菌生物农药研制及其应用技术研究
|
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。
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.
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。
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.
引文
[1]刘举鹏.浅谈我国一些重要代表性蝗虫.生物学通报. 1996, 31 (10) : 8～10.
[2] Greathead DJ. Biological control as a potential tool for locust and grasshopper control. In: Biological Control of Locusts and Grasshoppers. Lomer CJ & Prior C (eds.) CAB International. 1992, 4～7.
[3]马世骏.根治飞蝗灾害.科学通讯. 1956, 2: 52～56.
[4] Everts JW, Willemsen I, Stulp M, Simons L, Aukema B, Kammenga J. The toxic effects of deltamethrin on linyphiid and erigonid spiders in connection with ambient temperature, humidity and predation. Archives of Environmental Contamination and Toxicology, 1991, 20:20～24.
[5] Reichhardt T. Tight deadlines and data gaps fan fight on pesticide safety. Nature. 1998, 396: 207.
[6]马世骏.东亚飞蝗发生地的形成与改造.中国农业科学. 1960, 4: 18～22.
[7]陈永林.中国蝗虫灾害.见:中国自然灾害.孙广忠,王昂生,张丕远等著.学术书刊出版社. 1990, 235～250.
[8]张龙.蝗虫微孢子虫及其在蝗害治理中的作用.生物学通报. 1999, 34 (2) : 11～12.
[9]张龙,严毓骅,潘建梅等.蝗虫微孢子虫病在草原蝗虫优势种种群及空间的分布.昆虫学报. 1998, 41 (增刊).
[10] Henry JE, Oma EA. Pest control by Nosema locustae, a pathogen of grasshoppers and crickets. In : Burges HD. Edt . Microbial Control of Pests and Plant Diseases. Academic Press. 1981, 573～586.
[11]刘强.生物防治新思路:微孢子虫治蝗─访中国农业大学教授严毓骅.农民日报. 1998, 7月20日第二版.
[12]王丽英,严毓骅,董雁军.蝗虫微孢子虫对东亚飞蝗及蒙、新草原蝗虫的感染试验.北京农业大学学报. 1987, 13 (4): 495～462.
[13]王丽英,严毓骅,管致和.蝗虫微孢子虫对东亚飞蝗的实验感染.昆虫学报. 1990, 33 (1): 121～123.
[14]严毓骅.微孢子虫治蝗技术进展.见:全国生物防治学术讨论会论文集.北京:中国农科院生防所. 1991, 21～23.
[15]严毓骅.低耗持续治理蝗害新技术─微孢子虫治蝗.科技日报. 1995, 3428期:12月25日.
[16]刘晓建,李卫民,李淑君等.应用绿僵菌及其复合剂防治青海荒原蝗虫试验初报.青海草业. 2001, 10 (1): 13～15.
[17]邱式邦. 1993年非洲利用绿僵菌油剂防治蝗虫和蚱蜢的试验结果.生物防治通报. 1994, 10 (4): 186.
[18] Prior C, Greathead DJ. Biology control of locust: the potential for the exploitation of pathogens. FAO Plant Protection Bulletin. 1989, 37: 151～163.
[19] Lomer CJ, Bateman RP, Johnson DL, et al. Biological control of locusts and grasshoppers.Annu Rev Entomol. 2001, 46: 667~702.
[20] Abraham YJ, Bateman RP, Lomer C, Moore D, Paraiso A, Prior C. The IIBC-IITA-DFPV collaborative research programme on the biological control of locusts and grasshoppers. IOBC-WPRS Working Group "Insect Pathogens and Insect Parasitic Nematodes": 3rd European Meeting on Microbial Control of Pests. Edited by Smits PH. 1991, 56～62.
[21] Bateman RP, Douro-Kpindou OK, Kooyman C, Lomer C, Ouambama Z. Some observations on the dose transfer of mycoinsecticide sprays to desert locusts. Crop Protection. 1998, 17: 151～158.
[22] Price RE, Muller EJ, Brown HD, Uamba PD, Jone AA. The First Use of a Metarhizium flavoviride Mycoinsecticide for the Control of the Red Locust in a Recognised Outbreak Area. PPRI, South Africa. Science and Technology. 1997, 7: 357~363
[23] Sieglaff DH, Pereira RM, Capinera JL. Microbial control of Schistocerca americana (Orthoptera: A crididae ) by Metarhizium flavoviride (Deuteromycotina): instar dependent mortality and efficacy of ultra low volume application under greenhouse conditions. Journal of Economic Entomology. 1998, 91: 76～85.
[24] Milner RJ, Lim RP, Hunter DM. Risks to the aquatic ecosystem from the application of Metarhizium anisopliae for locust control in Australia. Pest Management Science. 2002, 58(7): 718～723.
[25] Moore D, Bateman RP, Carey M. Longterm storage of Metarhizium f lavoviride conidia in oil formulations for the control of locusts and grasshoppers. Biocontrol Science and Technology. 1995, 5 (2): 193～199.
[26] Lomer CJ, Batemsn RP, Godonou I, et al. Field infection of Zonocerus variegates following application of oil-based Formulation of Metarhizium flavoviride Conidia.Biocontrol Science and Techology. 1993, 3: 337～346.
[27] Hunter DM, Milner RJ and Spurgin PA. Aerial treatment of the Australian plague locust chortoicetes terminifera with metarhizium anisopliae. Bulletin of Entomological Research. 2001, 91(2): 93～99.
[28] Prior C. Susceptibility of target acridoids non-target acridoids to Metarhizium anisoplieaand M. flavoviride. In: K raull R P, Diallo D B, eds. New strategies in Locust control. Switzerland:Birkhauser Verlag Basel. 1997, 369～375.
[29] Bateman RP, Carey M, Moore D, Prior C. The enhance infectivity of Metarhizium flavoviride in oil formulation to desert locust at low humudities. Annals of applied biology. 1993, 122: 145～152.
[30] Bateman R. The development of a mycoinsecticide for the control of locusts and grasshoppers. International Institute of Biological control (UK). Outlook on Agriculture. 1997, 26(1): 13～18.
[31]魏文娟,任炳忠.我国蝗虫的生物防治技术及研究进展. 2002, 3(6):481～484.
[32]陆庆光,邓春光,陈长风.应用绿僵菌防治东亚飞蝗田间试验.昆虫天敌. 1996, 18 (4): 147～50.
[33]李保平,宋国庆,李国有.绿僵菌油剂防治荒漠草原蝗虫的田间试验.中国草地. 1999, 5:53～56.
[34]张泽华,高松,张刚应等.应用绿僵菌油剂防治内蒙草原蝗虫的效果.中国生物防治. 2000, 16(2): 49～52.
[35]陆庆光,邓春剩,张爱文等.四种不同绿僵菌菌株对东亚飞蝗毒力的初步观察.生物防治通报. 1993, 9(4):187.
[36]李保平, Roy Bateman,李国有等.绿僵菌油剂防治新疆山地草原蝗虫的田间试验.中国生物防治. 2000, 16(4): 145～147.
[37]樊美珍,郭超.中国虫生真菌研究与应用. 1993, 3:14～20.
[38] McCoy CW, Samson RA, Boucias DG. Entomogenous fungi. In: Ignoffo CM, Mandava NB. (Eds.), Handbook of Natural Pesticides: Microbial Insecticides, Vol. V, Part A, CRC Press, Inc., Boca Raton, Fl. 1988, pp151～236.
[39] Zimmermann G. The entomopathogenic fungus Metarhizium anisopliae and its potential as a biocontrol agent. Pesticide Science. 1993, 37:375~379.
[40] Tulloch M. The genus Metarhizium. Transaction of the British Mycological Society, 1976, 66: 407～411.
[41] Rombach MC, Aguda RM, Shepard BM, et al. Entomopathogenic fungi (Deuteromycotina) in the control of the black bug of rice, Scotinophara coarctata. Journal of Invertebrate Pathology. 1986, 48: 174～179.
[42]郭好礼,叶柏龄,岳莹玉等.绿僵菌属3个新种.真菌学报. 1986, 5(3): 177～184.
[43]刘爱英.绿僵菌属的新记录种.真菌学报. 1988, 7(3): 91～192.
[44]梁宗琦.虫草的无性型及其确定.西南农学报. 1991, 4(4): 1～8.
[45] Zacharuk RY. Fine structure of the fungus Metarhizium anisopliae infecting three species of larval Elateridae (Coleoptera). IV. Development within the host. Canadian Microbiology.1971, 17(4): 525~529.
[46] Fargues J. Etude des condition d’infection des larves poryphora, Leptinotarsa decemlieata say per Beauvera bassiana Entomopophaga. 1972, 17:319～337.
[47] Pendland JC, Heath M, Boucias DG. Function of a galactose-binding lectin from Spodoptera exigua larval hemolymph: opsonization of blastospores from entomogeneous hyphomycetes Journal of Insect Physiology. 1988, 34: 533～540.
[48] Boucias DG, Pendland JC, Latge JP. Nonspecific factors involved in attachment of enotomopathgenic deuteromycetes to host insect cuticle. Applied and Environmental Microbiology, 1988, 54: 1795～1805.
[49] St.Leger RJ, Donald W. Roberts and Richard C. Staples. Novel GTP-binding proteins in plasma membranes of the fungus Metarhizium anisopliae. Biochemical and Biophysical Research Communications. 1989, 164: 562～566.
[50]翟锦彬,黄秀梨,许萍.杀虫真菌-球孢白僵菌的昆虫致病机理研究究近况.微生物学通报. 1995, 22(1): 45～48.
[51] Hassan AE, Charnley AK. Ultrastructural study of the penetration by Metarhizium anisopliae through Dimilin-affected cuticle of Manduca sexta. Journal of Invertebrate Pathology, July 1989, 54(1): 117～124.
[52] Boucias DG, Pendland JC. In: Cole GT, Hoch HC, eds. The Fungal Spore and Disease Initiation in Plants and Animals. 1991, 101～127.
[53] Clarkson JM, Charnley AK. New insights into the mechanisms of fungal pathogenesis in insects. Trends Microbiol, 1996, 4: 197～203.
[54]李文华,王中康,殷幼平,夏玉先,彭国雄.理化因子对自僵菌附着胞形成的影响重庆大学学报. 2004, 27: 56～59.
[55] Neville AC. Cuticle:rganization in:Biology of the Integument.Invertebrates Berlin. 1984, 611～625.
[56] Charnley AK. and St. Leger RJ. The role of cuticle degrading enzymes in fungal pathogenesis of insects. In "The Fungal spore and Disease Initiation in plant and Animals" (G. T. Cole and H. C. Hoch, Eds.), 1991, pp267～287. Plenum, London.
[57]王荫长.昆虫生理生化.北京:中国农业出版社. 1994, 129～132.
[58] Charley AK. Physiological aspects of destructive pathogenesis in insects by fungi:A Speculative review. In "Invertebrate Microbial Interactions" (Anderson JM, Raynor ADM, and Walton DWH, Eds.), 1984, pp229～270. Cambridge. Press, Cambridge.
[59] St.Leger RJ, Lokesh J, Bidochka MJ, Roberts DW. Multiple Aminopeptidases Produced by Metarhizium anisopliae Journal of Invertebrate Pathology, 1995, 65(3): 313～314.
[60] Hassan AE and charnley AK. Combined effects of diflubenzuron and the entomopathogenic fungus on the tobacco hornworm Manduca sexta, Proc.10th Int. Congress. Plant Protection 1983, 3: 790.
[61] St. Leger RJ, Durrands PK, Charnley AK, Cooper RM. Role of extracellular chymoelastase in the virulence of Metarhizium anisopliae for Manduca sexta Journal of Invertebrate Pathology, 1988, 52(2): 285～293.
[62] Gupta SC, Leathers TD, E1-Sayed GN, Ignoff DM. Production of degradative enzymes by Metarhizium anisopliae during growth on defined media and insect cutide. Experimental Mycology. 1991, 15: 304～315.
[63] St.Leger RJ, Charnley AK, cooper RM. Cuticle-degrading enzymes of entomopathogenic fungi:Synthesis in culture on cuticle. Journal of Invertebrate Pathology. 1986, 48: 85～95.
[64] Micheal JB, George GK. Purification and Properties of an Extracellular Protease produced by the Entomopathogenic Fungus Beauveria bassiana. Applied and Environmental Microbiology. 1987, 48: 1679～1684.
[65] Smith RJ, and Grula EA. Chitinase is an inducible enzyme in Beauveria bassiana. Journal of Invertebrate Pathology. 1983, 15: 68～94.
[66] St.Leger RJ, Cooper RM, Charnley AK. Cuticle-degrading enzymes of entomopathogenic fungi: Regulation of production of chitinolytic emzymes. Journal of Gene Micobiology. 1986, 132:1509～1517.
[67] St.Leger RJ, Cooper RM, Charnley AK. Cuticle-degrading enzymes of entomopathogenic fungi:Cuticle degradation in vitro by enzymes from entomopathogen Journal of Invertebrate Pathology 1986, 47: 167～177.
[68]蒲蛰龙,李增智.昆虫真菌学.安徽科技出版社. 1996, 388～389页.
[69] Samuels RI, Reynolds SE, Charnley AK. Calcium channel activation of insect muscle by destruxins, insecticidal compounds produced by the entomopathogenic fungus Metarhizium anisopliae. Comparative Biochemistry and Physiology Part C: Comparative Pharmacology. 1988, 90 (2): 403～412.
[70]李农昌,樊美珍,朱玮,柴长宏.绿僵菌毒素的提取方法.林业实用技术. 1989, (10): 36～38.
[71] Dumas C, Matha V, Quiot JM. Effects of destruxins, cyclic depsipeptide mycotoxins,on calcium balance and phosphorylation of intracellular proteins in lepidopteran cell lines. Comparative Biochemistry and Physiology. 1996, 114(3): 213～219.
[72] Chen JW, Liu BL, Tzeng YM. Purification and quantification of destruxins A and destruxins B from Metarhizium anisopliae. Journal of Chromatography A. 1999, 830: 115～125.
[73] Kaijiang L and Roberts DW. The production of destruxins by the entomogenous fungus, Metarhizium anisopliae var major. Journal of Invertebrate Pathology, 1986, 47:120～122.
[74] Roberts DW. Toxins of entomopathogenic fungi. In: Microbial Control of Pests and Plant. Diseases 1970-1980, (H.D. Burges, ed). Academic Press, London, 1981, 441～464.
[75] St Leger RJ, Charnley AK, Cooper RM. Characterization of cuticle-degrading proteases produced by the entomopathogen Metarhizium anisopliae. Archives of Biochemistry and Biophysics. 1987, 253(1): 221～232.
[76] Smithson SL, Paterson IC, Bailey AM,Screen SE, Hunt BA, Cobb BD, Cooper RM, Charnley AK. and Clarkson JM. Cloning and characterization of a gene encoding a cuticle-degrading protease from the insect pathogenic fungus Metarhizium anisopliae. Gene. 1995, 166(1): 161～165.
[77] Bailey AM, Kershaw MJ, Hunt BA, Paterson IC, Charnley AK, et al. Cloning and sequence analysis of an intron-containing domain from a peptide synthetase-encoding gene of the entomopathogenic fungus Metarhizium anisopliae. Gene. 1996, 173: 195～197.
[78] Screen S, Bailey A, Charnley K, Cooper R, Clarkson J. Isolation of a nitrogen response regulator gene (nrr1) from Metarhizium anisopliae. Gene. 1998, 211: 17～24.
[79] Joshi L, St Leger RJ, Roberts DW. Isolation of a cDNA encoding a novel subtilisin-like protease (Pr1B) from the entomopathogenic fungus, Metarhizium anisopliae using differential display-RT-PCR. Gene. 1997(1～2): 1～8
[80] Xia YX, Gao MY, Clarkson JM, Charnley AK. Molecular cloning, characterisation, and expression of a neutral trehalase from the. insect pathogenic fungus Metarhizium anisopliae. Journal of Invertebrate Pathology. 2002, 80:127～137.
[81] Fang W, Bidochka MJ. Expression of genes involved in germination, conidiogenesis and pathogenesis in Metarhizium anisopliae using quantitative real-time RT-PCR. Mycological Research. 2006, 110: 1165～1171.
[82] Grell MN, Mouritzen P, Giese H. A Blumeria graminis gene family encoding proteins with a C-terminal variable region with homologues in pathogenic fungi. Gene. 2003, 311: 181～192.
[83] St.Leger RJ, Roberts DW. Engineering improved mycoinsecticides. Trend in Biotechnology 1997, 15: 83～85.
[84]张丽萍,程辉彩,王迎春等.绿僵菌固体生产条件的研究.农药. 2002, 41(7): 20～21.
[85]樊美珍,郭超,肖慧玲,胡茵.金龟子绿僵菌对几种林木害虫的致病性及防治应用试验.林业实用技术. 1989, 10: 25～27.
[86] Dubois T, Li Z, Jiafu H, Hajek AE. Efficacy of fiber bands impregnated with Beauveria brongniartii. cultures against Asian longhorned beetle, Anoplophora glabripennis (Coleoptera:Cerambycidae). Biological Control, 2004, 31: 320～328.
[87]宋漳.龟子绿僵菌液体深层培养研究.热带作物学报. 2002, 2: 35～38.
[88]李国栋.生物磁学.自然杂志. 1981, 7: 35～38.
[89]林韶湘.磁场对几种作物种子萌发的生物学效应.植物生理学通讯. 1983, 06: 41～43.
[90]宋漳.九株绿僵菌液体振荡培养初步研究[J]福建林学院学报. 1996, 1: 30～33.
[91]宋漳.应用磁化水深层培养绿僵菌液生分生孢子初探.林业科学. 2001, 2: 30～33.
[92] Anderson TE, Roberts WD. Compatibility of Beauveria bassiana isolates with insecticide formulations used in colorado potato beetle (Coleoptera: Chrysomelidae) control. Journal of Economic Entomology. 1983, 76: 1437-1441.
[93]宋漳.化学杀虫剂对绿僵菌的影响及菌药混用研究.福建林学院学报. 2001, 4: 37～40.
[94]刘晓建.应用绿僵菌及其复合剂防治青海草原蝗虫试验初报.青海草业. 2001, 1: 18～21.
[95] Lomer CJ, Bateman RJ, Godonou I, Kpindou D, Shah A, et al.,. Field infection of Zonocerus variegates following application of an oil based formulation of Metarhizium yavoviridae conidia. Biocontrol Science Technology. 1993, 3: 337-346
[96] Milner RJ. Metarhizium flavoviridae (F-I985) as a promising mycoinsecticide for Australian acridids. In: Microbial Control of Grasshoppers and Locusts. Goettel, M.S., Johnson, D.L. (Eds). Memoirs of the Entomological Society of Canada. 1997, 171: 287-300.
[97] Lomer CJ, Prior C, Kooyman C. Development of Metarhizium spp.for the control of grasshoppers and locusts. Memoirs of the Entomological Society of Canada. 1997, 171: 265～286.
[98] St. Leger RJ, Joshi L, Bidochka MJ, RIZZO NW, and Robters DW. Characterization and Ultrastructural Localization of Chitinases from Metarhizium anisopliae, M.flavoviride,and Beauveriabassiana during Fungal Invasion of Host (Manducasexta) Cuticle. Appliedand Environmental Microbiology. 1996, 62(3): 907～912.
[99] Delgado FX,James HB, Jerome AO, Will S. Field assessment of Beauveria bassiana (Badsamo) Vuillemin and potential synergism with diflubenzuron for control of savanna grasshopper complex (Orthoptera) in Mali. Journal of Invertebrate Pathology. 1999, 73(1): 34～39.
[100] Langewald J, Kooyman C, Douro-Kpindou, OK, Lomer C, Dahmoud AO, Mohamed HO. Field treatment of desert locust (Schistocerca gregaria Forskal) hoppers in the flield in Mauritania with an oil formulation of the entomopathogenic fungus Metarhizium flavoviride. Biocontrol Science and Technology. 1997, 7: 603-611.
[101] Kooyman C, Godonou I. Infection of Schistocerca gregaria (Orthoptera: Acrididae), hoppers by Metarhizium yavoviridae (Deuteromycotina: Hyphomycetes) conidia in an oil miscibleformulation applied under desert conditions. Bulletin of Entomological Research. 1997, 87: 105～107.
[102] Price RE, Bateman, RP, Brown HD, Butler ET, Muller EJ. Aerial spray trials against brown locust (Locustana pardalina,Walker) nymphs in South Africa using oil miscible formulations of Metarhizium yavoviridae. Crop Proeciont. 1997, 16: 345～51.
[103] Bateman RP, Price RE, Muller EJ, Brown HD. Controlling Brown locust hopper bands in South Africa with a myco-insecticide spray. Brighton, Crop Protection Conference -Pests and Diseases 6A-3. 1994, p609～616. BCPC.
[104]黄留玉. PCR最新技术原理、方法及运用.北京:化学工业出版社. 2005, 131～157.
[105] Xue B. Coodwin. PH, Annis SL. Pathotype identification of Leptophaeria maculans with PCR and oligonucleitide primers from ribosomal internal transcribed spacer sequences. molecular pathology. 1992, 41: 179～188.
[106]章晓波,徐洵.荧光探针研究新进展.生物工程进展. 2002, 20(2): 14～15.
[107] DNA/RNA Real-Time Quantitative PCR-Rev. B Applied Biosystems. http://www.appliedbiosystems.comcn/chinese/pe~h2~addl. html
[108] Morriso TB, Weis JJ, Wittwer CT. Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques. 1998, 24: 954~962.
[109]樊绮诗.定量PCR中常用参照系统.诊断学理论与实践. 2003, 2(3): 178～180.
[110] Bridge P, Spooner B. Soil fungi: diversity and detection.Plant Soil. 2001, 232: 147～154.
[111] van Elsas JD, Duarte GF, Keijzer-Wolters A, Smit E. Analysis of the dynamics of fungal communities in soil via fungal-specific PCR of soil DNA followed by denaturing gradient gel electrophoresis. Journal of Microbiological Methods. 2000, 43: 133～151.
[112] Raeymarkers L. Quantitative PCR. In: Lo, Y.M.D. (Ed.), Methods in Molecular Medicine: Clinical Applications of PCR.Humana Press, Totowa NJ, 1998, pp. 27～38.
[113] Heid CA, Stevens J, Livak KJ, Williams M. Real time quantitative PCR. Genome Res. 1996, 6: 986～994.
[114] Manju JD, Hester FW, Schaefer Ml. Detection of Cyclospora cayetanensis using a quantitative Real-tiom PCR assay. Journal of Microbiological Method. 2003, 1724: 1～10.
[115] Filion M, St-Arnaud M, Jabaji-Hare SH. Direct quantification of fungal DNA from soil substrate using real-time PCR. Journal of Microbiological Methods. 2003, 1735: 1～10
[116] Cullen DW, Lees AK, Toth IK, Duncan JM. Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR. Plant Pathology. 2002, 51: 281～292.
[117] Dwayne DH, George GK. Detection of the Entomopathgenic Fungus Beauveria bassiana within infected Migratory Grasshoppers (Melanoplus sanguinipes) using Polymerase Chain Reaction and Probe. Journal of invertebrate Pathology. 1996, 67: 21～27.
[119]唐启义.实用统计分析及其DPS数据处理系统.《北京:科学出版社》2002, P234～237.
[119]李智新,陈文海.非离子表面活性剂的HLB值.日用化学工业. 1990, 6: 41～44.
[120] Hunter DM, Milner RJ, Scanlan JC, Aerial treatment of the migratory locust, Locusta migratoria (L.) (Orthoptera: Acrididae) with Metarhizium anisopliae(Deuteromycotina: Hyphomycetes) in Australia. Crop Protection. 1999, 18: 699～704.
[121] Nguyen NT, Watt JW. The distribution and recovery of aerial ultra-low volume sprays for controlling nymphs of the Australian plague locust Chortoicetes terminifera Walker. Journal of Australian Entomological. 1981, 20: 269~275.
[122] Gillespie JP, Burnett C, Chamley AK. The immune response of the desert locust Schistocerca gregaria during mycosis of the entomopathogenic fungus, Metarhizium flavoviride. J Insect Physiol. 2000, 46: 429～437.
[123]单志萍,孟妤,姜文侯.丝状真菌三孢布拉霉DNA的提取研究.生物技术. 2001, 11(3): 5～6.
[124] Cooper SJB, Hewitt GM. Nuclear DNA sequence divergence between paratactic subspecies of the grasshopper Chorthippus parallelus . Insect Molecular Biology. 1993, 2(3): 185～194.
[125] Felice Driver, Rhichard J., et al., A taxonomic revision of Metarhium based on a phylogenetic analysis of rDNA sequence data. Mycology Research. 2000, 104(2): 134～150.
[126]张清平,彭国雄,王中康,殷幼,夏玉先.实时荧光定量PCR检测感病蝗虫体内绿僵菌rDNA基因.中国生物工程杂志. 2005, 25(11): 56～58.
[127] Manandhar JB. Effect of light , temperature and water potential on growth and sporulation of Microdochiumoryzea. Mycologia, 1998, 90: 995～1000.
[128]吴振强,彭景龙,李运南,张高贤.金龟子绿僵菌固态发酵环境变量优化研究农药. 2004, 43(3): 224.
[129] St. Leger RJ, Roberts D. Ambient pH is a major determinant in the expression of cuticle-degrating enzymes and hydrophobin by Metarhizium anisopliae. Applied and Environmental Microbiology. 1998, 64(2): 709～713.
[130] St.Leger RJ, Nelson JO, Screen SE. The entomopathogenic fungus Metarhizium anisopliae alters ambient pH, allowing extracellular protease production and activity. Microbiology. 1999, 145: 2691～2699.
[131] Chappell MA, Whitman DW. Thermoregulation. In A Biology of Grasshoppers, A. Joern and R. Chapman, eds. John Wiley & Sons, New York, 1990, pp 143～172.
[132] Mitchell DA, Von Meien OF. Mathematical modeling as a tool to investigate the design and operation of the zymotis packed-bed bioreactor for solid-state fermentation. Biotechnol Bioeng, 2000, 68: 127～135.
[133]许寿涛,应盛华,冯明光.十种常用农药与球孢白僵菌的生物学相容性.植物保护学报, 2002, 29(2): 158～162.
[134] Dillon RJ, and Charnley AK. Technique for accelerating and synchronising germination of conidia of the entomopathogenic fungus Metarhizium anisopliae. Archives of Microbiology. 1985, 142(2): 204～206.
[135] Batta YA. Production and testing of novel formulations of the entomopathogenic fungus Metarhizium anisopliae (Metschinkoff)Sorokin (Deuteromycotina: Hyphomycetes) Crop Protection. 2003, 22: 415～422.
[136] Peveling R, Attignon S, Langewald J, Ouambama Z. An assessment of the impact of biological and chemical grasshopper control agents on ground-dwelling arthropods in Niger, based on presence/absence sampling. Crop Proection. 1999, 18: 323～339.
[137] Thomas MB, Langewald J, Wood S N. Persistence of biopesticides and consequences for biological control of grasshopper and locusts. Pesticide Science. 1997, 49: 93～102.
[138] Cherry A, Jenkins N, Heviefo G, Bateman RP and Lomer C. A West African pilot scale production plant for aerial conidia of Metarhizium sp for use as a mycoinsecticide against locusts and grasshoppers. Biocontrol Science and Technology. 1999, 9: 35～51.
[139] Moore D, Reed M, Le Patourel G, Abraham YJ, Prior C. Reduction of feeding by the desert locust, Schistocerca gregaria, after infection with Metarhizium flavoviride. Journal of Invertebrate Pathology. 1992, 60: 304～307.
[140] Francisco P, Fernando EV, Stephen AR, Meredith B, Donald W, et al. Syspastospora parasitica, a mycoparasite of the fungus Beauveria bassiana attacking the Colorado potato beetle Leptinotarsa decemlineata: a tritrophic association. Journal of Insect Science. 2004, 4: 24.
[141] Flexner JL, Lighthart B, Croft BA. The effects of microbial pesticides on non-target, beneficial arthropods. Agricuture Ecosystem Environment. 1986, 16: 203～254.
[142] Smits JE, Johnson DL, Lomer CJ. Avian pathological and physiological responses to dietary exposure to the fungus Metarhizium flavoviride, an agent for control of grasshoppers and locusts in Africa. Journal of Wildlife Diseases. 1999, 35(2): 194～203.
[143]董辉,苏红田,高松,农向群,丛斌,张泽华.绿僵菌对蝗虫及其捕食性天敌的影响.中国生物防治. 2005, 21 (1): 60～62.
[144]蒲蛰龙,李增智.昆虫真菌学.合肥:安徽科技出版社, 1996, 246.
[145]沈萍,范秀容,李广武.微生物学实验第三版.北京:高等教育出版社. 1999, 90～92.
[146] Annapula G, Lut O. An overvicw of real-time PCR: Application to quantify cytokinc gene expression. Method. 2001, 21(1): 56～59.
[147] Higuchi R, Fockler C, et al. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology. 1993, 1(9): 1026~1030.
[148]王宁.定量PCR技术研究进展.国外医学临床生物化学与检验学分册. 2000, 21(6):314～316.
[149] Ke LD, Chen Z, Yung WK. A reliability test of standard-based quantitative PCR: exogenous vs endogenous standards. Molecular and Cellular Probes. 2000, 14(2): 127～135.
[2] Greathead DJ. Biological control as a potential tool for locust and grasshopper control. In: Biological Control of Locusts and Grasshoppers. Lomer CJ & Prior C (eds.) CAB International. 1992, 4～7.
[3]马世骏.根治飞蝗灾害.科学通讯. 1956, 2: 52～56.
[4] Everts JW, Willemsen I, Stulp M, Simons L, Aukema B, Kammenga J. The toxic effects of deltamethrin on linyphiid and erigonid spiders in connection with ambient temperature, humidity and predation. Archives of Environmental Contamination and Toxicology, 1991, 20:20～24.
[5] Reichhardt T. Tight deadlines and data gaps fan fight on pesticide safety. Nature. 1998, 396: 207.
[6]马世骏.东亚飞蝗发生地的形成与改造.中国农业科学. 1960, 4: 18～22.
[7]陈永林.中国蝗虫灾害.见:中国自然灾害.孙广忠,王昂生,张丕远等著.学术书刊出版社. 1990, 235～250.
[8]张龙.蝗虫微孢子虫及其在蝗害治理中的作用.生物学通报. 1999, 34 (2) : 11～12.
[9]张龙,严毓骅,潘建梅等.蝗虫微孢子虫病在草原蝗虫优势种种群及空间的分布.昆虫学报. 1998, 41 (增刊).
[10] Henry JE, Oma EA. Pest control by Nosema locustae, a pathogen of grasshoppers and crickets. In : Burges HD. Edt . Microbial Control of Pests and Plant Diseases. Academic Press. 1981, 573～586.
[11]刘强.生物防治新思路:微孢子虫治蝗─访中国农业大学教授严毓骅.农民日报. 1998, 7月20日第二版.
[12]王丽英,严毓骅,董雁军.蝗虫微孢子虫对东亚飞蝗及蒙、新草原蝗虫的感染试验.北京农业大学学报. 1987, 13 (4): 495～462.
[13]王丽英,严毓骅,管致和.蝗虫微孢子虫对东亚飞蝗的实验感染.昆虫学报. 1990, 33 (1): 121～123.
[14]严毓骅.微孢子虫治蝗技术进展.见:全国生物防治学术讨论会论文集.北京:中国农科院生防所. 1991, 21～23.
[15]严毓骅.低耗持续治理蝗害新技术─微孢子虫治蝗.科技日报. 1995, 3428期:12月25日.
[16]刘晓建,李卫民,李淑君等.应用绿僵菌及其复合剂防治青海荒原蝗虫试验初报.青海草业. 2001, 10 (1): 13～15.
[17]邱式邦. 1993年非洲利用绿僵菌油剂防治蝗虫和蚱蜢的试验结果.生物防治通报. 1994, 10 (4): 186.
[18] Prior C, Greathead DJ. Biology control of locust: the potential for the exploitation of pathogens. FAO Plant Protection Bulletin. 1989, 37: 151～163.
[19] Lomer CJ, Bateman RP, Johnson DL, et al. Biological control of locusts and grasshoppers.Annu Rev Entomol. 2001, 46: 667~702.
[20] Abraham YJ, Bateman RP, Lomer C, Moore D, Paraiso A, Prior C. The IIBC-IITA-DFPV collaborative research programme on the biological control of locusts and grasshoppers. IOBC-WPRS Working Group "Insect Pathogens and Insect Parasitic Nematodes": 3rd European Meeting on Microbial Control of Pests. Edited by Smits PH. 1991, 56～62.
[21] Bateman RP, Douro-Kpindou OK, Kooyman C, Lomer C, Ouambama Z. Some observations on the dose transfer of mycoinsecticide sprays to desert locusts. Crop Protection. 1998, 17: 151～158.
[22] Price RE, Muller EJ, Brown HD, Uamba PD, Jone AA. The First Use of a Metarhizium flavoviride Mycoinsecticide for the Control of the Red Locust in a Recognised Outbreak Area. PPRI, South Africa. Science and Technology. 1997, 7: 357~363
[23] Sieglaff DH, Pereira RM, Capinera JL. Microbial control of Schistocerca americana (Orthoptera: A crididae ) by Metarhizium flavoviride (Deuteromycotina): instar dependent mortality and efficacy of ultra low volume application under greenhouse conditions. Journal of Economic Entomology. 1998, 91: 76～85.
[24] Milner RJ, Lim RP, Hunter DM. Risks to the aquatic ecosystem from the application of Metarhizium anisopliae for locust control in Australia. Pest Management Science. 2002, 58(7): 718～723.
[25] Moore D, Bateman RP, Carey M. Longterm storage of Metarhizium f lavoviride conidia in oil formulations for the control of locusts and grasshoppers. Biocontrol Science and Technology. 1995, 5 (2): 193～199.
[26] Lomer CJ, Batemsn RP, Godonou I, et al. Field infection of Zonocerus variegates following application of oil-based Formulation of Metarhizium flavoviride Conidia.Biocontrol Science and Techology. 1993, 3: 337～346.
[27] Hunter DM, Milner RJ and Spurgin PA. Aerial treatment of the Australian plague locust chortoicetes terminifera with metarhizium anisopliae. Bulletin of Entomological Research. 2001, 91(2): 93～99.
[28] Prior C. Susceptibility of target acridoids non-target acridoids to Metarhizium anisoplieaand M. flavoviride. In: K raull R P, Diallo D B, eds. New strategies in Locust control. Switzerland:Birkhauser Verlag Basel. 1997, 369～375.
[29] Bateman RP, Carey M, Moore D, Prior C. The enhance infectivity of Metarhizium flavoviride in oil formulation to desert locust at low humudities. Annals of applied biology. 1993, 122: 145～152.
[30] Bateman R. The development of a mycoinsecticide for the control of locusts and grasshoppers. International Institute of Biological control (UK). Outlook on Agriculture. 1997, 26(1): 13～18.
[31]魏文娟,任炳忠.我国蝗虫的生物防治技术及研究进展. 2002, 3(6):481～484.
[32]陆庆光,邓春光,陈长风.应用绿僵菌防治东亚飞蝗田间试验.昆虫天敌. 1996, 18 (4): 147～50.
[33]李保平,宋国庆,李国有.绿僵菌油剂防治荒漠草原蝗虫的田间试验.中国草地. 1999, 5:53～56.
[34]张泽华,高松,张刚应等.应用绿僵菌油剂防治内蒙草原蝗虫的效果.中国生物防治. 2000, 16(2): 49～52.
[35]陆庆光,邓春剩,张爱文等.四种不同绿僵菌菌株对东亚飞蝗毒力的初步观察.生物防治通报. 1993, 9(4):187.
[36]李保平, Roy Bateman,李国有等.绿僵菌油剂防治新疆山地草原蝗虫的田间试验.中国生物防治. 2000, 16(4): 145～147.
[37]樊美珍,郭超.中国虫生真菌研究与应用. 1993, 3:14～20.
[38] McCoy CW, Samson RA, Boucias DG. Entomogenous fungi. In: Ignoffo CM, Mandava NB. (Eds.), Handbook of Natural Pesticides: Microbial Insecticides, Vol. V, Part A, CRC Press, Inc., Boca Raton, Fl. 1988, pp151～236.
[39] Zimmermann G. The entomopathogenic fungus Metarhizium anisopliae and its potential as a biocontrol agent. Pesticide Science. 1993, 37:375~379.
[40] Tulloch M. The genus Metarhizium. Transaction of the British Mycological Society, 1976, 66: 407～411.
[41] Rombach MC, Aguda RM, Shepard BM, et al. Entomopathogenic fungi (Deuteromycotina) in the control of the black bug of rice, Scotinophara coarctata. Journal of Invertebrate Pathology. 1986, 48: 174～179.
[42]郭好礼,叶柏龄,岳莹玉等.绿僵菌属3个新种.真菌学报. 1986, 5(3): 177～184.
[43]刘爱英.绿僵菌属的新记录种.真菌学报. 1988, 7(3): 91～192.
[44]梁宗琦.虫草的无性型及其确定.西南农学报. 1991, 4(4): 1～8.
[45] Zacharuk RY. Fine structure of the fungus Metarhizium anisopliae infecting three species of larval Elateridae (Coleoptera). IV. Development within the host. Canadian Microbiology.1971, 17(4): 525~529.
[46] Fargues J. Etude des condition d’infection des larves poryphora, Leptinotarsa decemlieata say per Beauvera bassiana Entomopophaga. 1972, 17:319～337.
[47] Pendland JC, Heath M, Boucias DG. Function of a galactose-binding lectin from Spodoptera exigua larval hemolymph: opsonization of blastospores from entomogeneous hyphomycetes Journal of Insect Physiology. 1988, 34: 533～540.
[48] Boucias DG, Pendland JC, Latge JP. Nonspecific factors involved in attachment of enotomopathgenic deuteromycetes to host insect cuticle. Applied and Environmental Microbiology, 1988, 54: 1795～1805.
[49] St.Leger RJ, Donald W. Roberts and Richard C. Staples. Novel GTP-binding proteins in plasma membranes of the fungus Metarhizium anisopliae. Biochemical and Biophysical Research Communications. 1989, 164: 562～566.
[50]翟锦彬,黄秀梨,许萍.杀虫真菌-球孢白僵菌的昆虫致病机理研究究近况.微生物学通报. 1995, 22(1): 45～48.
[51] Hassan AE, Charnley AK. Ultrastructural study of the penetration by Metarhizium anisopliae through Dimilin-affected cuticle of Manduca sexta. Journal of Invertebrate Pathology, July 1989, 54(1): 117～124.
[52] Boucias DG, Pendland JC. In: Cole GT, Hoch HC, eds. The Fungal Spore and Disease Initiation in Plants and Animals. 1991, 101～127.
[53] Clarkson JM, Charnley AK. New insights into the mechanisms of fungal pathogenesis in insects. Trends Microbiol, 1996, 4: 197～203.
[54]李文华,王中康,殷幼平,夏玉先,彭国雄.理化因子对自僵菌附着胞形成的影响重庆大学学报. 2004, 27: 56～59.
[55] Neville AC. Cuticle:rganization in:Biology of the Integument.Invertebrates Berlin. 1984, 611～625.
[56] Charnley AK. and St. Leger RJ. The role of cuticle degrading enzymes in fungal pathogenesis of insects. In "The Fungal spore and Disease Initiation in plant and Animals" (G. T. Cole and H. C. Hoch, Eds.), 1991, pp267～287. Plenum, London.
[57]王荫长.昆虫生理生化.北京:中国农业出版社. 1994, 129～132.
[58] Charley AK. Physiological aspects of destructive pathogenesis in insects by fungi:A Speculative review. In "Invertebrate Microbial Interactions" (Anderson JM, Raynor ADM, and Walton DWH, Eds.), 1984, pp229～270. Cambridge. Press, Cambridge.
[59] St.Leger RJ, Lokesh J, Bidochka MJ, Roberts DW. Multiple Aminopeptidases Produced by Metarhizium anisopliae Journal of Invertebrate Pathology, 1995, 65(3): 313～314.
[60] Hassan AE and charnley AK. Combined effects of diflubenzuron and the entomopathogenic fungus on the tobacco hornworm Manduca sexta, Proc.10th Int. Congress. Plant Protection 1983, 3: 790.
[61] St. Leger RJ, Durrands PK, Charnley AK, Cooper RM. Role of extracellular chymoelastase in the virulence of Metarhizium anisopliae for Manduca sexta Journal of Invertebrate Pathology, 1988, 52(2): 285～293.
[62] Gupta SC, Leathers TD, E1-Sayed GN, Ignoff DM. Production of degradative enzymes by Metarhizium anisopliae during growth on defined media and insect cutide. Experimental Mycology. 1991, 15: 304～315.
[63] St.Leger RJ, Charnley AK, cooper RM. Cuticle-degrading enzymes of entomopathogenic fungi:Synthesis in culture on cuticle. Journal of Invertebrate Pathology. 1986, 48: 85～95.
[64] Micheal JB, George GK. Purification and Properties of an Extracellular Protease produced by the Entomopathogenic Fungus Beauveria bassiana. Applied and Environmental Microbiology. 1987, 48: 1679～1684.
[65] Smith RJ, and Grula EA. Chitinase is an inducible enzyme in Beauveria bassiana. Journal of Invertebrate Pathology. 1983, 15: 68～94.
[66] St.Leger RJ, Cooper RM, Charnley AK. Cuticle-degrading enzymes of entomopathogenic fungi: Regulation of production of chitinolytic emzymes. Journal of Gene Micobiology. 1986, 132:1509～1517.
[67] St.Leger RJ, Cooper RM, Charnley AK. Cuticle-degrading enzymes of entomopathogenic fungi:Cuticle degradation in vitro by enzymes from entomopathogen Journal of Invertebrate Pathology 1986, 47: 167～177.
[68]蒲蛰龙,李增智.昆虫真菌学.安徽科技出版社. 1996, 388～389页.
[69] Samuels RI, Reynolds SE, Charnley AK. Calcium channel activation of insect muscle by destruxins, insecticidal compounds produced by the entomopathogenic fungus Metarhizium anisopliae. Comparative Biochemistry and Physiology Part C: Comparative Pharmacology. 1988, 90 (2): 403～412.
[70]李农昌,樊美珍,朱玮,柴长宏.绿僵菌毒素的提取方法.林业实用技术. 1989, (10): 36～38.
[71] Dumas C, Matha V, Quiot JM. Effects of destruxins, cyclic depsipeptide mycotoxins,on calcium balance and phosphorylation of intracellular proteins in lepidopteran cell lines. Comparative Biochemistry and Physiology. 1996, 114(3): 213～219.
[72] Chen JW, Liu BL, Tzeng YM. Purification and quantification of destruxins A and destruxins B from Metarhizium anisopliae. Journal of Chromatography A. 1999, 830: 115～125.
[73] Kaijiang L and Roberts DW. The production of destruxins by the entomogenous fungus, Metarhizium anisopliae var major. Journal of Invertebrate Pathology, 1986, 47:120～122.
[74] Roberts DW. Toxins of entomopathogenic fungi. In: Microbial Control of Pests and Plant. Diseases 1970-1980, (H.D. Burges, ed). Academic Press, London, 1981, 441～464.
[75] St Leger RJ, Charnley AK, Cooper RM. Characterization of cuticle-degrading proteases produced by the entomopathogen Metarhizium anisopliae. Archives of Biochemistry and Biophysics. 1987, 253(1): 221～232.
[76] Smithson SL, Paterson IC, Bailey AM,Screen SE, Hunt BA, Cobb BD, Cooper RM, Charnley AK. and Clarkson JM. Cloning and characterization of a gene encoding a cuticle-degrading protease from the insect pathogenic fungus Metarhizium anisopliae. Gene. 1995, 166(1): 161～165.
[77] Bailey AM, Kershaw MJ, Hunt BA, Paterson IC, Charnley AK, et al. Cloning and sequence analysis of an intron-containing domain from a peptide synthetase-encoding gene of the entomopathogenic fungus Metarhizium anisopliae. Gene. 1996, 173: 195～197.
[78] Screen S, Bailey A, Charnley K, Cooper R, Clarkson J. Isolation of a nitrogen response regulator gene (nrr1) from Metarhizium anisopliae. Gene. 1998, 211: 17～24.
[79] Joshi L, St Leger RJ, Roberts DW. Isolation of a cDNA encoding a novel subtilisin-like protease (Pr1B) from the entomopathogenic fungus, Metarhizium anisopliae using differential display-RT-PCR. Gene. 1997(1～2): 1～8
[80] Xia YX, Gao MY, Clarkson JM, Charnley AK. Molecular cloning, characterisation, and expression of a neutral trehalase from the. insect pathogenic fungus Metarhizium anisopliae. Journal of Invertebrate Pathology. 2002, 80:127～137.
[81] Fang W, Bidochka MJ. Expression of genes involved in germination, conidiogenesis and pathogenesis in Metarhizium anisopliae using quantitative real-time RT-PCR. Mycological Research. 2006, 110: 1165～1171.
[82] Grell MN, Mouritzen P, Giese H. A Blumeria graminis gene family encoding proteins with a C-terminal variable region with homologues in pathogenic fungi. Gene. 2003, 311: 181～192.
[83] St.Leger RJ, Roberts DW. Engineering improved mycoinsecticides. Trend in Biotechnology 1997, 15: 83～85.
[84]张丽萍,程辉彩,王迎春等.绿僵菌固体生产条件的研究.农药. 2002, 41(7): 20～21.
[85]樊美珍,郭超,肖慧玲,胡茵.金龟子绿僵菌对几种林木害虫的致病性及防治应用试验.林业实用技术. 1989, 10: 25～27.
[86] Dubois T, Li Z, Jiafu H, Hajek AE. Efficacy of fiber bands impregnated with Beauveria brongniartii. cultures against Asian longhorned beetle, Anoplophora glabripennis (Coleoptera:Cerambycidae). Biological Control, 2004, 31: 320～328.
[87]宋漳.龟子绿僵菌液体深层培养研究.热带作物学报. 2002, 2: 35～38.
[88]李国栋.生物磁学.自然杂志. 1981, 7: 35～38.
[89]林韶湘.磁场对几种作物种子萌发的生物学效应.植物生理学通讯. 1983, 06: 41～43.
[90]宋漳.九株绿僵菌液体振荡培养初步研究[J]福建林学院学报. 1996, 1: 30～33.
[91]宋漳.应用磁化水深层培养绿僵菌液生分生孢子初探.林业科学. 2001, 2: 30～33.
[92] Anderson TE, Roberts WD. Compatibility of Beauveria bassiana isolates with insecticide formulations used in colorado potato beetle (Coleoptera: Chrysomelidae) control. Journal of Economic Entomology. 1983, 76: 1437-1441.
[93]宋漳.化学杀虫剂对绿僵菌的影响及菌药混用研究.福建林学院学报. 2001, 4: 37～40.
[94]刘晓建.应用绿僵菌及其复合剂防治青海草原蝗虫试验初报.青海草业. 2001, 1: 18～21.
[95] Lomer CJ, Bateman RJ, Godonou I, Kpindou D, Shah A, et al.,. Field infection of Zonocerus variegates following application of an oil based formulation of Metarhizium yavoviridae conidia. Biocontrol Science Technology. 1993, 3: 337-346
[96] Milner RJ. Metarhizium flavoviridae (F-I985) as a promising mycoinsecticide for Australian acridids. In: Microbial Control of Grasshoppers and Locusts. Goettel, M.S., Johnson, D.L. (Eds). Memoirs of the Entomological Society of Canada. 1997, 171: 287-300.
[97] Lomer CJ, Prior C, Kooyman C. Development of Metarhizium spp.for the control of grasshoppers and locusts. Memoirs of the Entomological Society of Canada. 1997, 171: 265～286.
[98] St. Leger RJ, Joshi L, Bidochka MJ, RIZZO NW, and Robters DW. Characterization and Ultrastructural Localization of Chitinases from Metarhizium anisopliae, M.flavoviride,and Beauveriabassiana during Fungal Invasion of Host (Manducasexta) Cuticle. Appliedand Environmental Microbiology. 1996, 62(3): 907～912.
[99] Delgado FX,James HB, Jerome AO, Will S. Field assessment of Beauveria bassiana (Badsamo) Vuillemin and potential synergism with diflubenzuron for control of savanna grasshopper complex (Orthoptera) in Mali. Journal of Invertebrate Pathology. 1999, 73(1): 34～39.
[100] Langewald J, Kooyman C, Douro-Kpindou, OK, Lomer C, Dahmoud AO, Mohamed HO. Field treatment of desert locust (Schistocerca gregaria Forskal) hoppers in the flield in Mauritania with an oil formulation of the entomopathogenic fungus Metarhizium flavoviride. Biocontrol Science and Technology. 1997, 7: 603-611.
[101] Kooyman C, Godonou I. Infection of Schistocerca gregaria (Orthoptera: Acrididae), hoppers by Metarhizium yavoviridae (Deuteromycotina: Hyphomycetes) conidia in an oil miscibleformulation applied under desert conditions. Bulletin of Entomological Research. 1997, 87: 105～107.
[102] Price RE, Bateman, RP, Brown HD, Butler ET, Muller EJ. Aerial spray trials against brown locust (Locustana pardalina,Walker) nymphs in South Africa using oil miscible formulations of Metarhizium yavoviridae. Crop Proeciont. 1997, 16: 345～51.
[103] Bateman RP, Price RE, Muller EJ, Brown HD. Controlling Brown locust hopper bands in South Africa with a myco-insecticide spray. Brighton, Crop Protection Conference -Pests and Diseases 6A-3. 1994, p609～616. BCPC.
[104]黄留玉. PCR最新技术原理、方法及运用.北京:化学工业出版社. 2005, 131～157.
[105] Xue B. Coodwin. PH, Annis SL. Pathotype identification of Leptophaeria maculans with PCR and oligonucleitide primers from ribosomal internal transcribed spacer sequences. molecular pathology. 1992, 41: 179～188.
[106]章晓波,徐洵.荧光探针研究新进展.生物工程进展. 2002, 20(2): 14～15.
[107] DNA/RNA Real-Time Quantitative PCR-Rev. B Applied Biosystems. http://www.appliedbiosystems.comcn/chinese/pe~h2~addl. html
[108] Morriso TB, Weis JJ, Wittwer CT. Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques. 1998, 24: 954~962.
[109]樊绮诗.定量PCR中常用参照系统.诊断学理论与实践. 2003, 2(3): 178～180.
[110] Bridge P, Spooner B. Soil fungi: diversity and detection.Plant Soil. 2001, 232: 147～154.
[111] van Elsas JD, Duarte GF, Keijzer-Wolters A, Smit E. Analysis of the dynamics of fungal communities in soil via fungal-specific PCR of soil DNA followed by denaturing gradient gel electrophoresis. Journal of Microbiological Methods. 2000, 43: 133～151.
[112] Raeymarkers L. Quantitative PCR. In: Lo, Y.M.D. (Ed.), Methods in Molecular Medicine: Clinical Applications of PCR.Humana Press, Totowa NJ, 1998, pp. 27～38.
[113] Heid CA, Stevens J, Livak KJ, Williams M. Real time quantitative PCR. Genome Res. 1996, 6: 986～994.
[114] Manju JD, Hester FW, Schaefer Ml. Detection of Cyclospora cayetanensis using a quantitative Real-tiom PCR assay. Journal of Microbiological Method. 2003, 1724: 1～10.
[115] Filion M, St-Arnaud M, Jabaji-Hare SH. Direct quantification of fungal DNA from soil substrate using real-time PCR. Journal of Microbiological Methods. 2003, 1735: 1～10
[116] Cullen DW, Lees AK, Toth IK, Duncan JM. Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR. Plant Pathology. 2002, 51: 281～292.
[117] Dwayne DH, George GK. Detection of the Entomopathgenic Fungus Beauveria bassiana within infected Migratory Grasshoppers (Melanoplus sanguinipes) using Polymerase Chain Reaction and Probe. Journal of invertebrate Pathology. 1996, 67: 21～27.
[119]唐启义.实用统计分析及其DPS数据处理系统.《北京:科学出版社》2002, P234～237.
[119]李智新,陈文海.非离子表面活性剂的HLB值.日用化学工业. 1990, 6: 41～44.
[120] Hunter DM, Milner RJ, Scanlan JC, Aerial treatment of the migratory locust, Locusta migratoria (L.) (Orthoptera: Acrididae) with Metarhizium anisopliae(Deuteromycotina: Hyphomycetes) in Australia. Crop Protection. 1999, 18: 699～704.
[121] Nguyen NT, Watt JW. The distribution and recovery of aerial ultra-low volume sprays for controlling nymphs of the Australian plague locust Chortoicetes terminifera Walker. Journal of Australian Entomological. 1981, 20: 269~275.
[122] Gillespie JP, Burnett C, Chamley AK. The immune response of the desert locust Schistocerca gregaria during mycosis of the entomopathogenic fungus, Metarhizium flavoviride. J Insect Physiol. 2000, 46: 429～437.
[123]单志萍,孟妤,姜文侯.丝状真菌三孢布拉霉DNA的提取研究.生物技术. 2001, 11(3): 5～6.
[124] Cooper SJB, Hewitt GM. Nuclear DNA sequence divergence between paratactic subspecies of the grasshopper Chorthippus parallelus . Insect Molecular Biology. 1993, 2(3): 185～194.
[125] Felice Driver, Rhichard J., et al., A taxonomic revision of Metarhium based on a phylogenetic analysis of rDNA sequence data. Mycology Research. 2000, 104(2): 134～150.
[126]张清平,彭国雄,王中康,殷幼,夏玉先.实时荧光定量PCR检测感病蝗虫体内绿僵菌rDNA基因.中国生物工程杂志. 2005, 25(11): 56～58.
[127] Manandhar JB. Effect of light , temperature and water potential on growth and sporulation of Microdochiumoryzea. Mycologia, 1998, 90: 995～1000.
[128]吴振强,彭景龙,李运南,张高贤.金龟子绿僵菌固态发酵环境变量优化研究农药. 2004, 43(3): 224.
[129] St. Leger RJ, Roberts D. Ambient pH is a major determinant in the expression of cuticle-degrating enzymes and hydrophobin by Metarhizium anisopliae. Applied and Environmental Microbiology. 1998, 64(2): 709～713.
[130] St.Leger RJ, Nelson JO, Screen SE. The entomopathogenic fungus Metarhizium anisopliae alters ambient pH, allowing extracellular protease production and activity. Microbiology. 1999, 145: 2691～2699.
[131] Chappell MA, Whitman DW. Thermoregulation. In A Biology of Grasshoppers, A. Joern and R. Chapman, eds. John Wiley & Sons, New York, 1990, pp 143～172.
[132] Mitchell DA, Von Meien OF. Mathematical modeling as a tool to investigate the design and operation of the zymotis packed-bed bioreactor for solid-state fermentation. Biotechnol Bioeng, 2000, 68: 127～135.
[133]许寿涛,应盛华,冯明光.十种常用农药与球孢白僵菌的生物学相容性.植物保护学报, 2002, 29(2): 158～162.
[134] Dillon RJ, and Charnley AK. Technique for accelerating and synchronising germination of conidia of the entomopathogenic fungus Metarhizium anisopliae. Archives of Microbiology. 1985, 142(2): 204～206.
[135] Batta YA. Production and testing of novel formulations of the entomopathogenic fungus Metarhizium anisopliae (Metschinkoff)Sorokin (Deuteromycotina: Hyphomycetes) Crop Protection. 2003, 22: 415～422.
[136] Peveling R, Attignon S, Langewald J, Ouambama Z. An assessment of the impact of biological and chemical grasshopper control agents on ground-dwelling arthropods in Niger, based on presence/absence sampling. Crop Proection. 1999, 18: 323～339.
[137] Thomas MB, Langewald J, Wood S N. Persistence of biopesticides and consequences for biological control of grasshopper and locusts. Pesticide Science. 1997, 49: 93～102.
[138] Cherry A, Jenkins N, Heviefo G, Bateman RP and Lomer C. A West African pilot scale production plant for aerial conidia of Metarhizium sp for use as a mycoinsecticide against locusts and grasshoppers. Biocontrol Science and Technology. 1999, 9: 35～51.
[139] Moore D, Reed M, Le Patourel G, Abraham YJ, Prior C. Reduction of feeding by the desert locust, Schistocerca gregaria, after infection with Metarhizium flavoviride. Journal of Invertebrate Pathology. 1992, 60: 304～307.
[140] Francisco P, Fernando EV, Stephen AR, Meredith B, Donald W, et al. Syspastospora parasitica, a mycoparasite of the fungus Beauveria bassiana attacking the Colorado potato beetle Leptinotarsa decemlineata: a tritrophic association. Journal of Insect Science. 2004, 4: 24.
[141] Flexner JL, Lighthart B, Croft BA. The effects of microbial pesticides on non-target, beneficial arthropods. Agricuture Ecosystem Environment. 1986, 16: 203～254.
[142] Smits JE, Johnson DL, Lomer CJ. Avian pathological and physiological responses to dietary exposure to the fungus Metarhizium flavoviride, an agent for control of grasshoppers and locusts in Africa. Journal of Wildlife Diseases. 1999, 35(2): 194～203.
[143]董辉,苏红田,高松,农向群,丛斌,张泽华.绿僵菌对蝗虫及其捕食性天敌的影响.中国生物防治. 2005, 21 (1): 60～62.
[144]蒲蛰龙,李增智.昆虫真菌学.合肥:安徽科技出版社, 1996, 246.
[145]沈萍,范秀容,李广武.微生物学实验第三版.北京:高等教育出版社. 1999, 90～92.
[146] Annapula G, Lut O. An overvicw of real-time PCR: Application to quantify cytokinc gene expression. Method. 2001, 21(1): 56～59.
[147] Higuchi R, Fockler C, et al. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology. 1993, 1(9): 1026~1030.
[148]王宁.定量PCR技术研究进展.国外医学临床生物化学与检验学分册. 2000, 21(6):314～316.
[149] Ke LD, Chen Z, Yung WK. A reliability test of standard-based quantitative PCR: exogenous vs endogenous standards. Molecular and Cellular Probes. 2000, 14(2): 127～135.