球孢白僵菌HFW-05对粉虱、棉铃虫侵染方式及高温胁迫法提高菌株耐热力研究
|
摘要
球孢白僵菌(Beauveria bassiana (Balsamo)Vuillemin)是目前国内外应用最广泛的昆虫病原真菌之一,能够侵染15个目149个科的700多种昆虫,并可在田间形成昆虫流行病,防治效果持久。HFW-05菌株是河北省农科院植保所杀虫微生物实验室分离筛选出的,对小菜峨、粉虱具有很高的杀虫活性,在前期的工作基础上,本研究扩大了该菌株的杀虫谱,明确了HFW-05菌株对烟粉虱和棉铃虫的不同侵染方式,通过合适的高温胁迫处理方法,在一定程度上提高了HFW-05菌株对较高温度的耐受力,并得到了耐热性状较稳定的新菌株。
通过对烟粉虱、小菜蛾、棉铃虫、东亚飞蝗和蛴螬类(华北大黑鳃金龟、暗黑鳃金龟和铜绿丽金龟)进行室内生物测定,其中烟粉虱采用浸渍法,小菜蛾采用喷雾法,棉铃虫采用浸渍和饲喂两种方法,东亚飞蝗采用背板点滴法,蛴螬采用拌土法,表明HFW-05菌株对以上害虫均有毒力,除棉铃虫外,其余供试昆虫均可由表皮侵染,其中对烟粉虱和小菜蛾的效果最好,LC50分别为4.20×105孢子/mL和1.59×106孢子/mL,且田间小区试验防效均可达80%;HFW-05菌株对其他几种昆虫的作用时间较长,以浓度为107孢子/mL的孢悬液接种,9d时东亚飞蝗2龄蝗蝻的死亡率为62.67%;蛴螬类害虫中,对14d铜绿异丽金龟、12d华北大黑鳃金龟和9d暗黑鳃金龟幼虫的校正死亡率分别为60%、46%和40.74%。对于棉铃虫的胃毒作用较明显,6d后的校正死亡率为72%,且对试虫的体重和取食量有显著的抑制作用,但通过表皮侵染效果不佳。
组织学研究表明,该菌株对烟粉虱主要从体壁接触侵染,菌丝优先从腹部侵入,也最早从腹部穿出体表;但未观察到从体壁侵入棉铃虫的现象,而通过胃毒侵染在消化道内发现分生孢子,并萌发生长至体内充满菌丝时导致试虫死亡。已报道的白僵菌对棉铃虫的侵染方式多为体壁侵染,这一现象进一步说明白僵菌对寄主侵入方式因昆虫的种类、虫态、环境条件等不同而有差别。
为了提高HFW-05菌株对较高温度环境的适应性,采用高温胁迫的方法对HFW-05菌株进行了定向驯化。选用48℃下分别水浴处理30、40、50、60、70、80、90、100、110、120min,以孢子残存率、产孢量、生长速率、萌发中时、对小菜蛾的毒力以及产生的胞外蛋白酶和几丁质酶的活性为指标,将胁迫后一代中耐热性较好的处理以相同的处理方式连续胁迫至第8代,通过比较各测定指标,结果表明在48℃下处理60和110min并连续胁迫至第8代时菌种的耐热性最好,萌发率、产孢量及在较高温度环境下对小菜蛾的毒力均显著高于对照菌株。将筛选出的耐热菌株和对照菌株同时在30℃下继代培养,5个世代后30℃下对照菌株的萌发率仅为15%,而耐热菌株的萌发率均达50%;对照菌株对小菜蛾致死率极低,耐热菌株在30℃和模拟田间环境的条件下对小菜蛾校正死亡率均在50%以上,32℃下也有30%以上的校正死亡率。本研究表明,合适的热处理方式可以在较大程度上提高白僵菌的耐热性,为解决白僵菌在田间高温环境下应用具有局限性的问题提供了科学依据。
Beauveria bassiana (Balsamo) Vuillemin is one of the entomopathogenic fungi which applied the most twidely presently at home and abroad, to pest control effeetively.I tcould infect more than 700 Species insects of 149 Families in 15 Orders, and caused the epidemicdis of pest, lasted control. A more virulent strain, HFW-05, to nymph of B. tabaci and P. xylostella was obtained by Laboratory of Microbial insecticide Institute of Plant Protection, Hebei Agricultural and Forestry Science. In this paper, the expansion of insecticidal spectrum of the HFW-05 strain, and infection of Bemisia tabaci and Plutella xylostella would be studied. The thermotolerance of the strain could be improved by high temperature stress, and got a new more stable strain.
The HFW-05 strain has virulence against B. tabaci, P. xylostella, Helicoverpa armigera, Locusta migratoria manilensis, Holotrichia oblira, Holotrichia parallela and Anomala corpulenta was showed by the bioassay. All of those pest could be infected through the skin, except Helicoverpa armigera. The formulation brought on high mortality of two tested Pest in the lab and field. HFW-05 strain was toxic to the 3rd larvae of Bemisia tabaci with an LC50 of 4.20×105conidia/mL, to the 2rd of Plutella xylostella with an LC50 of 1.59×106 conidia/mL, and the mortality could be 80% in the field. The action time to the other pests would be longer .The mortality of HFW-05 with 107 conidia/mL to locusta migratoria manilensis after 9 days and to Holotrichia oblira, Holotrichia parallela and Anomala corpulenta after 12 days could be 62.67%, 60%, 46% and 40.74%. Stomach infection had be found on Helicoverpa armigera, Weight and food consumption bad be inhibited obviously.
The paraffin slice of and Helicoverpa armigera infected by HFW-05 showed that the conidia infected Bemisia tabaci through the skin, hypha first penetrated the abdomen,and firstly pierced surface from the abdomen . Skin infection was not found on Helicoverpa armigera, conidias were found in digestive tract,germination and growth was showed ,and tissues was full of hypha when the pest was dead. Skin infection on Helicoverpa armigera by B. bassiana had been reported mostly. Further explanation of the phenomenon is the infection to the host by Beauveria bassiana is different for differernt insect species, instars and environmental conditions.
HFW-05 strain was domesticated directionaly with high temperature stress for improving the thermotolerance. Conidia suspension were exposed to a thermal stress of water bath under 48°C for 30, 40, 50, 60, 70, 80, 90, 100, 110, 120min, then mensurated the residue viabilities of conidia, rate of growth, sporulation, hydrophobicity virulence, extracellular protease and chitinase. The treatment which have high heat resistance after thermal stress were teratde with the same approach to the 8th generation.The results showed that thermotolerance of the strain which were exposed to a thermal stress of under 48°C water bath for 60 and 110 min to the 8th generation have been obviously improved.The heat-resistant and control strains were subcultured at the same time at 30℃. After 5 generation, the germination rate of the control strain was 15%,but the heat-resistant strains, were 50%, the Virulence against Plutella xylostella by the control was too little ,but the heat-resistant strains, were more than 50% at 30℃and more than 30% at 32℃. This study shows that the appropriate heat treatment can greatly improve the heat resistance of Beauveria bassiana,it provided a scientific basis for solving the problem that the high-temperature environment limitations Beauveria bassiana faced when applicated in the field.
通过对烟粉虱、小菜蛾、棉铃虫、东亚飞蝗和蛴螬类(华北大黑鳃金龟、暗黑鳃金龟和铜绿丽金龟)进行室内生物测定,其中烟粉虱采用浸渍法,小菜蛾采用喷雾法,棉铃虫采用浸渍和饲喂两种方法,东亚飞蝗采用背板点滴法,蛴螬采用拌土法,表明HFW-05菌株对以上害虫均有毒力,除棉铃虫外,其余供试昆虫均可由表皮侵染,其中对烟粉虱和小菜蛾的效果最好,LC50分别为4.20×105孢子/mL和1.59×106孢子/mL,且田间小区试验防效均可达80%;HFW-05菌株对其他几种昆虫的作用时间较长,以浓度为107孢子/mL的孢悬液接种,9d时东亚飞蝗2龄蝗蝻的死亡率为62.67%;蛴螬类害虫中,对14d铜绿异丽金龟、12d华北大黑鳃金龟和9d暗黑鳃金龟幼虫的校正死亡率分别为60%、46%和40.74%。对于棉铃虫的胃毒作用较明显,6d后的校正死亡率为72%,且对试虫的体重和取食量有显著的抑制作用,但通过表皮侵染效果不佳。
组织学研究表明,该菌株对烟粉虱主要从体壁接触侵染,菌丝优先从腹部侵入,也最早从腹部穿出体表;但未观察到从体壁侵入棉铃虫的现象,而通过胃毒侵染在消化道内发现分生孢子,并萌发生长至体内充满菌丝时导致试虫死亡。已报道的白僵菌对棉铃虫的侵染方式多为体壁侵染,这一现象进一步说明白僵菌对寄主侵入方式因昆虫的种类、虫态、环境条件等不同而有差别。
为了提高HFW-05菌株对较高温度环境的适应性,采用高温胁迫的方法对HFW-05菌株进行了定向驯化。选用48℃下分别水浴处理30、40、50、60、70、80、90、100、110、120min,以孢子残存率、产孢量、生长速率、萌发中时、对小菜蛾的毒力以及产生的胞外蛋白酶和几丁质酶的活性为指标,将胁迫后一代中耐热性较好的处理以相同的处理方式连续胁迫至第8代,通过比较各测定指标,结果表明在48℃下处理60和110min并连续胁迫至第8代时菌种的耐热性最好,萌发率、产孢量及在较高温度环境下对小菜蛾的毒力均显著高于对照菌株。将筛选出的耐热菌株和对照菌株同时在30℃下继代培养,5个世代后30℃下对照菌株的萌发率仅为15%,而耐热菌株的萌发率均达50%;对照菌株对小菜蛾致死率极低,耐热菌株在30℃和模拟田间环境的条件下对小菜蛾校正死亡率均在50%以上,32℃下也有30%以上的校正死亡率。本研究表明,合适的热处理方式可以在较大程度上提高白僵菌的耐热性,为解决白僵菌在田间高温环境下应用具有局限性的问题提供了科学依据。
Beauveria bassiana (Balsamo) Vuillemin is one of the entomopathogenic fungi which applied the most twidely presently at home and abroad, to pest control effeetively.I tcould infect more than 700 Species insects of 149 Families in 15 Orders, and caused the epidemicdis of pest, lasted control. A more virulent strain, HFW-05, to nymph of B. tabaci and P. xylostella was obtained by Laboratory of Microbial insecticide Institute of Plant Protection, Hebei Agricultural and Forestry Science. In this paper, the expansion of insecticidal spectrum of the HFW-05 strain, and infection of Bemisia tabaci and Plutella xylostella would be studied. The thermotolerance of the strain could be improved by high temperature stress, and got a new more stable strain.
The HFW-05 strain has virulence against B. tabaci, P. xylostella, Helicoverpa armigera, Locusta migratoria manilensis, Holotrichia oblira, Holotrichia parallela and Anomala corpulenta was showed by the bioassay. All of those pest could be infected through the skin, except Helicoverpa armigera. The formulation brought on high mortality of two tested Pest in the lab and field. HFW-05 strain was toxic to the 3rd larvae of Bemisia tabaci with an LC50 of 4.20×105conidia/mL, to the 2rd of Plutella xylostella with an LC50 of 1.59×106 conidia/mL, and the mortality could be 80% in the field. The action time to the other pests would be longer .The mortality of HFW-05 with 107 conidia/mL to locusta migratoria manilensis after 9 days and to Holotrichia oblira, Holotrichia parallela and Anomala corpulenta after 12 days could be 62.67%, 60%, 46% and 40.74%. Stomach infection had be found on Helicoverpa armigera, Weight and food consumption bad be inhibited obviously.
The paraffin slice of and Helicoverpa armigera infected by HFW-05 showed that the conidia infected Bemisia tabaci through the skin, hypha first penetrated the abdomen,and firstly pierced surface from the abdomen . Skin infection was not found on Helicoverpa armigera, conidias were found in digestive tract,germination and growth was showed ,and tissues was full of hypha when the pest was dead. Skin infection on Helicoverpa armigera by B. bassiana had been reported mostly. Further explanation of the phenomenon is the infection to the host by Beauveria bassiana is different for differernt insect species, instars and environmental conditions.
HFW-05 strain was domesticated directionaly with high temperature stress for improving the thermotolerance. Conidia suspension were exposed to a thermal stress of water bath under 48°C for 30, 40, 50, 60, 70, 80, 90, 100, 110, 120min, then mensurated the residue viabilities of conidia, rate of growth, sporulation, hydrophobicity virulence, extracellular protease and chitinase. The treatment which have high heat resistance after thermal stress were teratde with the same approach to the 8th generation.The results showed that thermotolerance of the strain which were exposed to a thermal stress of under 48°C water bath for 60 and 110 min to the 8th generation have been obviously improved.The heat-resistant and control strains were subcultured at the same time at 30℃. After 5 generation, the germination rate of the control strain was 15%,but the heat-resistant strains, were 50%, the Virulence against Plutella xylostella by the control was too little ,but the heat-resistant strains, were more than 50% at 30℃and more than 30% at 32℃. This study shows that the appropriate heat treatment can greatly improve the heat resistance of Beauveria bassiana,it provided a scientific basis for solving the problem that the high-temperature environment limitations Beauveria bassiana faced when applicated in the field.
引文
[1]李荣森,罗绍彬.微生物防治害虫[M].北京:科技出版社, 1983. 27-45.
[2]刘健,陈洪章,李佐虎.白僵菌杀虫剂生产工艺研究状况与展望[J].中国生物防治, 2003, l9(2): 86-90.
[3] St Leger R J, Screen S. Prospects for strain improvement of fungal pathogens of insects and weeds. Fungal as Biocontrol Agents[M]. London: CABI Publish House, 2001: 219-237.
[4] Blake R,Bextun E, Harlan G, et al. Field applications of bait-formulated Beauveria bassiana alginate pellets for biological control of the red imported fire ant [J]. Biol Control,2002,31(4): 746-752.
[5] Felipe T, Mario Z,Raquela, et al. Pathogenicity of Beauveria bassiana (deuteromycota: hyphomycetes) against the cactus weevil, Metamasius spinolae (coleoptera: curculionidae) under laboratory conditions[J]. Florida ntomol, 2004, 87(4): 533-539.
[6]蒲蛰龙.昆虫病理学[M].广州:广东科技出版社, 1994. 346-350.
[7]蒲蛰龙,李增智.昆虫真菌学[M].合肥:安徽科学技术出版社, 1996. 297一302.
[8] Clarkson J M,Charnley A K. New insights into the mechanisms of fungal pathogenesis in insects[J]. Trends in Microbiology, 1996, 4: 197-203.
[9]宋入梅,王云滨,李瑞军,等.甜菜夜蛾感染白僵菌、绿僵菌后的病症及组织病理学变化[J].华北农学报, 2006, 21(增刊): 171-174.
[10]刘召,雷仲仁,花保祯,等.烟粉虱感染白僵菌后的组织病理变化[J].植物保护, 2006, 32(3): 52-54.
[11]王晓红,黄大庄,杨忠岐,等.白僵菌感染桑天牛幼虫致病过程的显微观察[J].蚕业科学, 2009, 35(2): 374-378.
[12]林华峰,李增智,胡萃.不同环境中松毛虫感染白僵菌过程的组织病理变化[J].安徽农业大学学报, 1998, 25(4): 330-335.
[13]季香云,杨长举.白僵菌的致病性与应用[J].中国生物防治, 2003, 19(2): 82-85.
[14]胡景江,樊美珍.球孢白僵菌胞外蛋白酶与其毒力的关系[J].安徽农业大学学报, 1996, 23(3): 273-278.
[15]姚剑,李增智,樊美珍.虫生真菌入侵过程中酶的作用[J].安徽农业大学学报, 1996, 23(3): 308-308.
[16]杨怀文.我国农业病虫害生物防治进展—迈入21世纪的中国生物防治[C].北京:中国农业科技出版社, 2005: 1-5.
[17]徐均焕,冯明光,许谦.昆虫病原真菌胞外蛋白酶的病理作用与测定技术[J].中国生物防治, 1998, 14(3): 123-129.
[18] St. Leger R L, CooPer R M, ChamLey A K. Produetion of cuticle-degrading enzymes by the entomopathogen M.anisoplia during infection of cuticles from Calliphora vomitoria sexta[J]. Gen Microbiol, 1987, 133: 1371-1382.
[19] Michael J, Bidochka, Geogre G Knaehatour et al. Regulation of extracellular protease in the entomopathogenic fungus B.bassiana[J]. Experi Myeol, 1988, 12: 161-168.
[20]冯明光.胞外蛋白酶和脂酶活性作为球孢白僵菌毒力指标的可靠性分析[J].微生物学报, 1998, 38(6): 461-467.
[21]胡锦江,樊美珍.不同世代白僵菌胞外蛋白酶及其与毒力的关系[J].西北林学院学报(自然版), 1993, 8(4): 36-40.
[22]樊美珍,胡锦江,李农昌,等.球孢白僵菌胞外蛋白酶及其与毒力关系的研究[J].微生物学通报, 1994, 21(4): 202-206.
[23]姚剑,黄大庆.球孢白僵菌蛋白质酶、几丁质酶和β-N-乙酰葡萄糖苷酶产生水平及其与毒力关系的研究[J].宿州学院学报, 2004, 19(4): 102-106.
[24]刘智辉.球孢白僵菌对亚洲玉米螟毒力检测的研究[D].武汉:华中农业大学, 2005: 43-45.
[25] Fang W G, Leng B, Xiao Y H, et al. Cloning of Beauveria bassiana chitinase gene Bbchitl and its application to improve fungal strain virolence[J]. Appl. Environ. Microbiol. 2005, 71(1): 363-370.
[26]范艳华.球孢白僵菌降解寄主体壁的几丁质酶和蛋白酶的分子改良[D].重庆:西南大学, 2006: 4-6.
[27] St Leger R J, Bidochka M J, Roberts D W. Isoforms of the cuticle-degrading Pr1 proteinase and production of a metallo proteinase by Metarhizium anisopliae[J]. Arch.Biochem. Biophys, 1994, 313(1): 1-7.
[28]金洁,张作法,时连根,等.昆虫病原白僵菌的分子生物学研究进展[J].科技通报, 2007, 23(6): 842-846.
[29]林海萍,魏锦瑜,毛胜凤,等.球孢白僵菌蛋白酶、几丁质酶、脂肪酶活性与其毒力相关性[J].中国生物防治, 2008, 24(3): 290-292.
[30] Iskandarov U S, Guzalova A G, Davranov K D. Effects of nutrient medium composition and temperature on the germination of conidia and the entomopathogenic activity of the fungi[J]. Prikl Biokhim Mikrobiol, 2006, 42(1): 81-85.
[31]武觑文.微生物杀虫剂的应用—松毛虫综合管理[M].北京:中国林业出版社, 1990: 256-273.
[32]邝灼彬,吕利华,冯夏,等.温度及常见农药对球孢白僵菌生物学特性的影响[J].华南农业大学学报, 2005, 26(3): 26-29.
[33] Lingg A J, Donaldson M D. Biotic and abiotic factors affecting stability of Beauveria bassiana conidia in soil[J]. Journal of Invertebrate Pathology, 1981, 38(2): 197-200.
[34]何恒果.高毒效杀蚜球孢白僵菌菌株筛选[J].中国生态农业学报, 2007, 15(3): 113-116.
[35] He Y R, Lu L H, Kuang Z B, et al. Effect of temperature and humidity on the virulence of beetle-derived Beauveria bassiana (Balsamo) Vuillemin (Deuteromycetes: Moniliales) against the daikon leaf beetle, Phaedon brassicae Baly (Coleoptera: Chrysomelidae)[J]. Acta Entomologica Sinica, 2005, 48(5): 679-686.
[36] Bello D, Padin G, Juarez S, et al. Biocontrol of Acanthoscelides obtectus and Sitophilus oryzae with diatomaceous earth and Beauveria bassiana on stored grains[J]. Biocontrol science and technology. 2006, 16(1-2): 215-220.
[37] Vassilakos T N, Athanassiou C G,Kavallieratos N G, et al. Influence of temperature on the insecticidal effect of Beauveria bassiana in combination with diatomaceous earth against Rhyzopertha dominica and Sitophilus oryzae on stored wheat[J]. Biological Control, 2006, 38(2): 270-281.
[38]农向群,高松,邓春生,李俊国,等.白僵菌绿僵菌分生孢子对高温的耐受力[J].中国生物防治, 1999, 15(3): 111-114.
[39]俞佳,冯明光.基于分生孢子热胁迫反应的球孢白僵菌耐热菌株筛选[J].菌物学报, 2006, 25(3): 278-283.
[40]王成树,王四宝,樊美珍,等.球孢白僵菌菌株耐热力与贮藏稳定性的关系[J].中国生物防治, 1999, 15(4): 162-165.
[41]刘银泉,冯明光,刘树生,等.不同温度下球孢白僵菌对桃蚜的毒力[J].中国生物防治, 2000, 16(2): 56-60.
[42]孙鲁娟,吴孔明,郭予元.不同温、湿度下白僵菌对棉铃虫幼虫的致病力[J].昆虫学报, 2004, 44(4): 501-506.
[43]应盛华.球孢白僵菌孢壁蛋白相关的耐热分子机理及其耐热性状的遗传改良[D].杭州:浙江大学, 2006.
[44]秦长生.保水剂对白僵菌发芽率影响的初步研究[J].广东林业科技, 2003, 19(4): 30-32.
[45] Lazzarini G M J, Rocha L F N, Luz C. Impact of moisture on in vitro germination of Metarhizium anisopliae and Beauveria bassiana and their activity on Triatoma infestans[J]. Mycological Researeh, 2006, 11(4): 485-492.
[46]王滨,樊美珍,李增智.球孢白僵菌选择性培养基的筛选[J].安徽农业大学学报, 2000, 27(l): 23-28.
[47]李农昌,樊美珍,李春如.白僵菌有关培养条件及其与毒力关系的研究[J].安徽农业大学学报, 1996, 23(3): 254-259.
[48]黄长春,汤坚,王成树.紫外辐射对球孢白僵菌的影响及适宜保护剂的选择[J].安徽农业大学学报, 1996, 23(3): 346-350.
[49] Leger S T, Durrands P K, Charnley A K, et al. The role of extracellular chymoelastase in the virulence of Metarhizium anisopliae for Manduca sexta[J].Journal of Invertebrate Pathology,1988,52:85-294.
[50] Leger S T,Frank DC,Roberts DW,et al.Molecular cloning and regulatory analysis of the cuticle-degrading protease structural gene from the entomopathogenic fungus Meaarhizium anisopliae[J]. Eur J Biochem, 1992, 204: 991-1001.
[51] Leger S T. The role of cuticle-degrading protease in fungal pathogenesis of insects[J]. Can J Bot, 1995, 73: 1119-1125.
[52] Leger S T, Joshi L, Bidochka M J, et al. Construction of an improved mycoinsecticide overexpressing a toxic protease[J]. Proc. Natl. Acad. Sci. USA. 1996, 93: 6349-6354.
[53] Leger S T, Robert DW. Engineering improved mycoinsecticides[J]. Trend Biotechnol, 1997, 15: 83-85.
[54] Joshi L, St Leger R J, Bidochka M J. Cloning of a cuticle degrading protease from the entomopathogenic fungus, Beauveria bassiana[J]. FEMS Microbiol Lett, 1995, 125(23): 211-217.
[55]方卫国,张永军,杨星勇,等.球孢白僵菌降解昆虫体壁蛋白酶CDEP-1的克隆与序列分析[J].遗传学报, 2002, 29(3): 278-282.
[56]张永军,彭国雄,方卫国,等.球孢白僵菌胞外蛋白酶及类枯草杆菌蛋白酶的诱导[J].应用与环境生物学报, 2000, 6(2): 182-186.
[57]贺小红,曾智,付祖姣,等.杀虫球孢白僵菌菌株筛选及类枯草杆菌蛋白酶基因的克隆[J].湖南师范大学自然科学学报, 2008, 31(1): 100-103.
[58] Fang W G, Leng B, Xiao Y H, et al. Cloning of Beauveria bassiana chitinase gene Bbchitl and its application to improve fungal strain virolence[J]. Appl. Environ. Microbiol. 2005, 71(1): 363-370.
[59]冯静.几丁酶-蛋白酶融合基因的构建与球孢白僵菌毒力的提高[D].重庆:西南大学, 2006: 34-38.
[60] Fan Y, Fang W, Xiao Y, et al. Directed evolution for in-creased chitinase activity[J]. Appl Microbiol Biotechnol. 2007, 76(1): 135-139.
[61]郭书娟.利用家蚕几丁质酶Bmchi提高球孢白僵菌的毒力[D].重庆:西南大学, 2007: 38-41.
[62]曹伟平,王金耀,王容燕,等.球孢白僵菌HFW-05的诱变筛选及其对烟粉虱若虫的毒力测定[J].中国生物防治, 2007, 23(2): 133-137.
[63] White J. Silver leaf whitefly extends range[J]. Califomia Agriculture, 1998, (52): 6-7.
[64] Feng M G, Poprawski T J, Khachatourians G G. Production,formulation and application of the entomopathgenic fungus Beauveria bassiana for insect control: currentstatus[J]. Biocontrol Sci Technol, 1994, 4: 3-34.
[65] Poprawski T J. Control of Bemisia Argentifolii on collards using different formulations and rates of Beauveria bassiana[J]. Arthropod Manage, 1999, (24): 122-123.
[66] Feng M G, Chen B, Ying SH. Trials of Beauveria bassiana, Paecilomyces fumosoroseus and imidacloprid for management of Trialeurodes vaporariorum (Homoptera: Aleyrodidae) on greenhouse grown lettuce[J]. Biocontrol Science and Technology, 2004, 14(6): 531-54.
[67]邓春生,张爱文,农向群,等.卵孢白僵菌对花生蛴螬的田间防治效果[J].中国生物防治1995, 11(2): 56-59.
[68]刘召,雷仲仁,花保祯,等.烟粉虱感染白僵菌后的组织病理变化[J].植物保护, 2006, 32(3): 52-54
[69] Pekrul S, Grula E A. Mode of infection of the corn earworm (Heliothis zea) by Beauveria bassianaas revealed by scanning microscopy[J]. J.Invertebr. Pathol., 1979, 34: 238-247.
[70]孙鲁娟,吴孔明.棉铃虫感染白僵菌后的组织病理变化[J].中国生物防, 2000, 16(3): 102-106.
[71]翟锦彬,黄秀梨.球孢白僵菌在棉铃虫表皮上萌发研究[J].微生物学报, 1997, 37(2): 54-158.
[72]王海川,尤民生.绿僵菌对昆虫的入侵机理[J].微生物学通报, 1999, 26(1): 71-73.
[73] Stleger R J, Frank D C, Roberts D M, et al. Molecular cloning and regulatory analysis of the cuticle-degrading protease structural gene from the entomopathogenic fungus, Metarhiziumanisopliae[J]. Eur J Biochem, 1992, 204: 991-1001.
[74]樊美珍,胡景江.白僵菌酯酶同工酶、脂肪酸与其毒力的关系[J].安徽农业大学学报, 1996, 23(3): 260-266.
[75]孙钰,郝建欣,丛威,等.磁性微球吸附法研究盐藻细胞的疏水性[J].海洋科学, 2003, 27(2): 37-40.
[76] Lowry O H, Rosbrough N J, Farr A L, et al. Protein measurement with folin phenol reagent[J]. Journal of biological chemistry, 1951, 193: 265-275.
[77] Mauch F, Hadwiger L, Boller T.Ethylene: Symptom,Not Signal for the Induction of Chitinase andβ-1, 3-Glucanase in Pea Pods by Pathogens and Elicitors[J]. Plant Physiol, 1984, 76: 607-611.
[78]陈新芳,田沈,王菊,等.提高木糖和葡萄糖乙醇产率的菌种驯化[J].太阳能学报, 2005, 26(2): 215-218.
[79]李慧,王异静.菌种驯化技术在红曲发酵产品中的应用[J].食品基础科学, 2006(2): 126-128.
[80]惠丰立,魏明卉,刘宗才,等.酸花生奶生产菌种驯化的研究[J].南都学坛(自然科学版), 2000, 20(6): 37-38.
[81]陶玉贵,颜世雷,潘军,等.氧化乐果降解真菌的筛选及其特性研究[J]. 2007, 26(5): 1744-1748.
[82]谢翎,陈红梅,蒲顺昌,等.实时荧光定量PCR检测球孢白僵菌热休克蛋白基因hsp70在几种胁迫条件下的表达[J].菌物学报, 2009, 28(6): 806-812.
[83]黄惠芳,马飞.热激蛋白的分子进化研究[J].厦门大学学报(自然科学版), 2004, 43(8): 166-170.
[84] Nover L, Scharf K D. Heat stress proteins and transcription factors[J]. Cellular and Molecular Life Sciences, 1997, 53(1): 80-103.
[85]李晓鲁,彭毅志.热激蛋白70与热激反应[J].生命的化学, 2002, 22(2): 145-147.
[86]李冰祥,蔡惠罗,陈永林.昆虫的热休克反应和热休克蛋白[J].昆虫学报, 1997, 40(4): 417-427.
[87] Deane E E, Won N Y. Cloning and characterization of the hsp70 mutigene family from silver sea bream: modulated gene expression between warm and cold temperature acclimation[J]. Biochemical and Biophysical Research ommunications. 2005, 330(3): 776-783.
[88]谢翎,陈红梅,蒲顺昌,等.球孢白僵菌热休克蛋白基因Bbhsp70的cDNA及上游序列克隆与分析[J].菌物学报, 2009, 28(2): 283-288.
[89]唐晓庆,樊美珍,李增智.球孢白僵菌继代培养中菌落局变现象及环境影响因素的研究[J].真菌学报, 1996, 15(3 ): 188-I96.
[90]唐晓庆,樊美珍,李增智.白僵菌菌种退化及其控制[J].安徽农业大学学报, 1996, 23(3): 239-245.
[91]蔡国贵,林庆源,徐耀昌,等.白僵菌菌株退化与培养条件关系及其控制技术[J].福建林学院学报, 2001, 21 (1): 76-79.
[2]刘健,陈洪章,李佐虎.白僵菌杀虫剂生产工艺研究状况与展望[J].中国生物防治, 2003, l9(2): 86-90.
[3] St Leger R J, Screen S. Prospects for strain improvement of fungal pathogens of insects and weeds. Fungal as Biocontrol Agents[M]. London: CABI Publish House, 2001: 219-237.
[4] Blake R,Bextun E, Harlan G, et al. Field applications of bait-formulated Beauveria bassiana alginate pellets for biological control of the red imported fire ant [J]. Biol Control,2002,31(4): 746-752.
[5] Felipe T, Mario Z,Raquela, et al. Pathogenicity of Beauveria bassiana (deuteromycota: hyphomycetes) against the cactus weevil, Metamasius spinolae (coleoptera: curculionidae) under laboratory conditions[J]. Florida ntomol, 2004, 87(4): 533-539.
[6]蒲蛰龙.昆虫病理学[M].广州:广东科技出版社, 1994. 346-350.
[7]蒲蛰龙,李增智.昆虫真菌学[M].合肥:安徽科学技术出版社, 1996. 297一302.
[8] Clarkson J M,Charnley A K. New insights into the mechanisms of fungal pathogenesis in insects[J]. Trends in Microbiology, 1996, 4: 197-203.
[9]宋入梅,王云滨,李瑞军,等.甜菜夜蛾感染白僵菌、绿僵菌后的病症及组织病理学变化[J].华北农学报, 2006, 21(增刊): 171-174.
[10]刘召,雷仲仁,花保祯,等.烟粉虱感染白僵菌后的组织病理变化[J].植物保护, 2006, 32(3): 52-54.
[11]王晓红,黄大庄,杨忠岐,等.白僵菌感染桑天牛幼虫致病过程的显微观察[J].蚕业科学, 2009, 35(2): 374-378.
[12]林华峰,李增智,胡萃.不同环境中松毛虫感染白僵菌过程的组织病理变化[J].安徽农业大学学报, 1998, 25(4): 330-335.
[13]季香云,杨长举.白僵菌的致病性与应用[J].中国生物防治, 2003, 19(2): 82-85.
[14]胡景江,樊美珍.球孢白僵菌胞外蛋白酶与其毒力的关系[J].安徽农业大学学报, 1996, 23(3): 273-278.
[15]姚剑,李增智,樊美珍.虫生真菌入侵过程中酶的作用[J].安徽农业大学学报, 1996, 23(3): 308-308.
[16]杨怀文.我国农业病虫害生物防治进展—迈入21世纪的中国生物防治[C].北京:中国农业科技出版社, 2005: 1-5.
[17]徐均焕,冯明光,许谦.昆虫病原真菌胞外蛋白酶的病理作用与测定技术[J].中国生物防治, 1998, 14(3): 123-129.
[18] St. Leger R L, CooPer R M, ChamLey A K. Produetion of cuticle-degrading enzymes by the entomopathogen M.anisoplia during infection of cuticles from Calliphora vomitoria sexta[J]. Gen Microbiol, 1987, 133: 1371-1382.
[19] Michael J, Bidochka, Geogre G Knaehatour et al. Regulation of extracellular protease in the entomopathogenic fungus B.bassiana[J]. Experi Myeol, 1988, 12: 161-168.
[20]冯明光.胞外蛋白酶和脂酶活性作为球孢白僵菌毒力指标的可靠性分析[J].微生物学报, 1998, 38(6): 461-467.
[21]胡锦江,樊美珍.不同世代白僵菌胞外蛋白酶及其与毒力的关系[J].西北林学院学报(自然版), 1993, 8(4): 36-40.
[22]樊美珍,胡锦江,李农昌,等.球孢白僵菌胞外蛋白酶及其与毒力关系的研究[J].微生物学通报, 1994, 21(4): 202-206.
[23]姚剑,黄大庆.球孢白僵菌蛋白质酶、几丁质酶和β-N-乙酰葡萄糖苷酶产生水平及其与毒力关系的研究[J].宿州学院学报, 2004, 19(4): 102-106.
[24]刘智辉.球孢白僵菌对亚洲玉米螟毒力检测的研究[D].武汉:华中农业大学, 2005: 43-45.
[25] Fang W G, Leng B, Xiao Y H, et al. Cloning of Beauveria bassiana chitinase gene Bbchitl and its application to improve fungal strain virolence[J]. Appl. Environ. Microbiol. 2005, 71(1): 363-370.
[26]范艳华.球孢白僵菌降解寄主体壁的几丁质酶和蛋白酶的分子改良[D].重庆:西南大学, 2006: 4-6.
[27] St Leger R J, Bidochka M J, Roberts D W. Isoforms of the cuticle-degrading Pr1 proteinase and production of a metallo proteinase by Metarhizium anisopliae[J]. Arch.Biochem. Biophys, 1994, 313(1): 1-7.
[28]金洁,张作法,时连根,等.昆虫病原白僵菌的分子生物学研究进展[J].科技通报, 2007, 23(6): 842-846.
[29]林海萍,魏锦瑜,毛胜凤,等.球孢白僵菌蛋白酶、几丁质酶、脂肪酶活性与其毒力相关性[J].中国生物防治, 2008, 24(3): 290-292.
[30] Iskandarov U S, Guzalova A G, Davranov K D. Effects of nutrient medium composition and temperature on the germination of conidia and the entomopathogenic activity of the fungi[J]. Prikl Biokhim Mikrobiol, 2006, 42(1): 81-85.
[31]武觑文.微生物杀虫剂的应用—松毛虫综合管理[M].北京:中国林业出版社, 1990: 256-273.
[32]邝灼彬,吕利华,冯夏,等.温度及常见农药对球孢白僵菌生物学特性的影响[J].华南农业大学学报, 2005, 26(3): 26-29.
[33] Lingg A J, Donaldson M D. Biotic and abiotic factors affecting stability of Beauveria bassiana conidia in soil[J]. Journal of Invertebrate Pathology, 1981, 38(2): 197-200.
[34]何恒果.高毒效杀蚜球孢白僵菌菌株筛选[J].中国生态农业学报, 2007, 15(3): 113-116.
[35] He Y R, Lu L H, Kuang Z B, et al. Effect of temperature and humidity on the virulence of beetle-derived Beauveria bassiana (Balsamo) Vuillemin (Deuteromycetes: Moniliales) against the daikon leaf beetle, Phaedon brassicae Baly (Coleoptera: Chrysomelidae)[J]. Acta Entomologica Sinica, 2005, 48(5): 679-686.
[36] Bello D, Padin G, Juarez S, et al. Biocontrol of Acanthoscelides obtectus and Sitophilus oryzae with diatomaceous earth and Beauveria bassiana on stored grains[J]. Biocontrol science and technology. 2006, 16(1-2): 215-220.
[37] Vassilakos T N, Athanassiou C G,Kavallieratos N G, et al. Influence of temperature on the insecticidal effect of Beauveria bassiana in combination with diatomaceous earth against Rhyzopertha dominica and Sitophilus oryzae on stored wheat[J]. Biological Control, 2006, 38(2): 270-281.
[38]农向群,高松,邓春生,李俊国,等.白僵菌绿僵菌分生孢子对高温的耐受力[J].中国生物防治, 1999, 15(3): 111-114.
[39]俞佳,冯明光.基于分生孢子热胁迫反应的球孢白僵菌耐热菌株筛选[J].菌物学报, 2006, 25(3): 278-283.
[40]王成树,王四宝,樊美珍,等.球孢白僵菌菌株耐热力与贮藏稳定性的关系[J].中国生物防治, 1999, 15(4): 162-165.
[41]刘银泉,冯明光,刘树生,等.不同温度下球孢白僵菌对桃蚜的毒力[J].中国生物防治, 2000, 16(2): 56-60.
[42]孙鲁娟,吴孔明,郭予元.不同温、湿度下白僵菌对棉铃虫幼虫的致病力[J].昆虫学报, 2004, 44(4): 501-506.
[43]应盛华.球孢白僵菌孢壁蛋白相关的耐热分子机理及其耐热性状的遗传改良[D].杭州:浙江大学, 2006.
[44]秦长生.保水剂对白僵菌发芽率影响的初步研究[J].广东林业科技, 2003, 19(4): 30-32.
[45] Lazzarini G M J, Rocha L F N, Luz C. Impact of moisture on in vitro germination of Metarhizium anisopliae and Beauveria bassiana and their activity on Triatoma infestans[J]. Mycological Researeh, 2006, 11(4): 485-492.
[46]王滨,樊美珍,李增智.球孢白僵菌选择性培养基的筛选[J].安徽农业大学学报, 2000, 27(l): 23-28.
[47]李农昌,樊美珍,李春如.白僵菌有关培养条件及其与毒力关系的研究[J].安徽农业大学学报, 1996, 23(3): 254-259.
[48]黄长春,汤坚,王成树.紫外辐射对球孢白僵菌的影响及适宜保护剂的选择[J].安徽农业大学学报, 1996, 23(3): 346-350.
[49] Leger S T, Durrands P K, Charnley A K, et al. The role of extracellular chymoelastase in the virulence of Metarhizium anisopliae for Manduca sexta[J].Journal of Invertebrate Pathology,1988,52:85-294.
[50] Leger S T,Frank DC,Roberts DW,et al.Molecular cloning and regulatory analysis of the cuticle-degrading protease structural gene from the entomopathogenic fungus Meaarhizium anisopliae[J]. Eur J Biochem, 1992, 204: 991-1001.
[51] Leger S T. The role of cuticle-degrading protease in fungal pathogenesis of insects[J]. Can J Bot, 1995, 73: 1119-1125.
[52] Leger S T, Joshi L, Bidochka M J, et al. Construction of an improved mycoinsecticide overexpressing a toxic protease[J]. Proc. Natl. Acad. Sci. USA. 1996, 93: 6349-6354.
[53] Leger S T, Robert DW. Engineering improved mycoinsecticides[J]. Trend Biotechnol, 1997, 15: 83-85.
[54] Joshi L, St Leger R J, Bidochka M J. Cloning of a cuticle degrading protease from the entomopathogenic fungus, Beauveria bassiana[J]. FEMS Microbiol Lett, 1995, 125(23): 211-217.
[55]方卫国,张永军,杨星勇,等.球孢白僵菌降解昆虫体壁蛋白酶CDEP-1的克隆与序列分析[J].遗传学报, 2002, 29(3): 278-282.
[56]张永军,彭国雄,方卫国,等.球孢白僵菌胞外蛋白酶及类枯草杆菌蛋白酶的诱导[J].应用与环境生物学报, 2000, 6(2): 182-186.
[57]贺小红,曾智,付祖姣,等.杀虫球孢白僵菌菌株筛选及类枯草杆菌蛋白酶基因的克隆[J].湖南师范大学自然科学学报, 2008, 31(1): 100-103.
[58] Fang W G, Leng B, Xiao Y H, et al. Cloning of Beauveria bassiana chitinase gene Bbchitl and its application to improve fungal strain virolence[J]. Appl. Environ. Microbiol. 2005, 71(1): 363-370.
[59]冯静.几丁酶-蛋白酶融合基因的构建与球孢白僵菌毒力的提高[D].重庆:西南大学, 2006: 34-38.
[60] Fan Y, Fang W, Xiao Y, et al. Directed evolution for in-creased chitinase activity[J]. Appl Microbiol Biotechnol. 2007, 76(1): 135-139.
[61]郭书娟.利用家蚕几丁质酶Bmchi提高球孢白僵菌的毒力[D].重庆:西南大学, 2007: 38-41.
[62]曹伟平,王金耀,王容燕,等.球孢白僵菌HFW-05的诱变筛选及其对烟粉虱若虫的毒力测定[J].中国生物防治, 2007, 23(2): 133-137.
[63] White J. Silver leaf whitefly extends range[J]. Califomia Agriculture, 1998, (52): 6-7.
[64] Feng M G, Poprawski T J, Khachatourians G G. Production,formulation and application of the entomopathgenic fungus Beauveria bassiana for insect control: currentstatus[J]. Biocontrol Sci Technol, 1994, 4: 3-34.
[65] Poprawski T J. Control of Bemisia Argentifolii on collards using different formulations and rates of Beauveria bassiana[J]. Arthropod Manage, 1999, (24): 122-123.
[66] Feng M G, Chen B, Ying SH. Trials of Beauveria bassiana, Paecilomyces fumosoroseus and imidacloprid for management of Trialeurodes vaporariorum (Homoptera: Aleyrodidae) on greenhouse grown lettuce[J]. Biocontrol Science and Technology, 2004, 14(6): 531-54.
[67]邓春生,张爱文,农向群,等.卵孢白僵菌对花生蛴螬的田间防治效果[J].中国生物防治1995, 11(2): 56-59.
[68]刘召,雷仲仁,花保祯,等.烟粉虱感染白僵菌后的组织病理变化[J].植物保护, 2006, 32(3): 52-54
[69] Pekrul S, Grula E A. Mode of infection of the corn earworm (Heliothis zea) by Beauveria bassianaas revealed by scanning microscopy[J]. J.Invertebr. Pathol., 1979, 34: 238-247.
[70]孙鲁娟,吴孔明.棉铃虫感染白僵菌后的组织病理变化[J].中国生物防, 2000, 16(3): 102-106.
[71]翟锦彬,黄秀梨.球孢白僵菌在棉铃虫表皮上萌发研究[J].微生物学报, 1997, 37(2): 54-158.
[72]王海川,尤民生.绿僵菌对昆虫的入侵机理[J].微生物学通报, 1999, 26(1): 71-73.
[73] Stleger R J, Frank D C, Roberts D M, et al. Molecular cloning and regulatory analysis of the cuticle-degrading protease structural gene from the entomopathogenic fungus, Metarhiziumanisopliae[J]. Eur J Biochem, 1992, 204: 991-1001.
[74]樊美珍,胡景江.白僵菌酯酶同工酶、脂肪酸与其毒力的关系[J].安徽农业大学学报, 1996, 23(3): 260-266.
[75]孙钰,郝建欣,丛威,等.磁性微球吸附法研究盐藻细胞的疏水性[J].海洋科学, 2003, 27(2): 37-40.
[76] Lowry O H, Rosbrough N J, Farr A L, et al. Protein measurement with folin phenol reagent[J]. Journal of biological chemistry, 1951, 193: 265-275.
[77] Mauch F, Hadwiger L, Boller T.Ethylene: Symptom,Not Signal for the Induction of Chitinase andβ-1, 3-Glucanase in Pea Pods by Pathogens and Elicitors[J]. Plant Physiol, 1984, 76: 607-611.
[78]陈新芳,田沈,王菊,等.提高木糖和葡萄糖乙醇产率的菌种驯化[J].太阳能学报, 2005, 26(2): 215-218.
[79]李慧,王异静.菌种驯化技术在红曲发酵产品中的应用[J].食品基础科学, 2006(2): 126-128.
[80]惠丰立,魏明卉,刘宗才,等.酸花生奶生产菌种驯化的研究[J].南都学坛(自然科学版), 2000, 20(6): 37-38.
[81]陶玉贵,颜世雷,潘军,等.氧化乐果降解真菌的筛选及其特性研究[J]. 2007, 26(5): 1744-1748.
[82]谢翎,陈红梅,蒲顺昌,等.实时荧光定量PCR检测球孢白僵菌热休克蛋白基因hsp70在几种胁迫条件下的表达[J].菌物学报, 2009, 28(6): 806-812.
[83]黄惠芳,马飞.热激蛋白的分子进化研究[J].厦门大学学报(自然科学版), 2004, 43(8): 166-170.
[84] Nover L, Scharf K D. Heat stress proteins and transcription factors[J]. Cellular and Molecular Life Sciences, 1997, 53(1): 80-103.
[85]李晓鲁,彭毅志.热激蛋白70与热激反应[J].生命的化学, 2002, 22(2): 145-147.
[86]李冰祥,蔡惠罗,陈永林.昆虫的热休克反应和热休克蛋白[J].昆虫学报, 1997, 40(4): 417-427.
[87] Deane E E, Won N Y. Cloning and characterization of the hsp70 mutigene family from silver sea bream: modulated gene expression between warm and cold temperature acclimation[J]. Biochemical and Biophysical Research ommunications. 2005, 330(3): 776-783.
[88]谢翎,陈红梅,蒲顺昌,等.球孢白僵菌热休克蛋白基因Bbhsp70的cDNA及上游序列克隆与分析[J].菌物学报, 2009, 28(2): 283-288.
[89]唐晓庆,樊美珍,李增智.球孢白僵菌继代培养中菌落局变现象及环境影响因素的研究[J].真菌学报, 1996, 15(3 ): 188-I96.
[90]唐晓庆,樊美珍,李增智.白僵菌菌种退化及其控制[J].安徽农业大学学报, 1996, 23(3): 239-245.
[91]蔡国贵,林庆源,徐耀昌,等.白僵菌菌株退化与培养条件关系及其控制技术[J].福建林学院学报, 2001, 21 (1): 76-79.