绿僵菌对蜱致病因子的分析和初步应用
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摘要
蜱隶属于蜱螨目、蜱总科,寄生于动物体表,广泛分布于我国各省区,是严重危害畜牧业生产的一类外寄生虫,不仅可通过叮咬吸血导致动物生产性能的下降,还可以传播动物和人的多种传染病(包括病毒病和细菌病)和寄生虫病(如:泰勒虫病和巴贝斯虫病),给畜牧业造成严重经济损失。目前对蜱的防制方式主要以化学药物防治为主,但随着蜱耐药性的产生以及人们对环境污染和药物残留等公共卫生问题的关注,该方法的应用在一定程度上受到限制,人们越来越将目光转移到免疫预防和生物防治等绿色控制方法上来。
致病性病原真菌作为一种很有潜力的生物控制因子,已在农业害虫生物防治领域得到广泛地应用,而在兽医线虫和外寄生虫病防治的研究才刚刚起步。本文以对蜱具有高致病力的菌株为研究对象,研究其毒力因子——胞外蛋白酶和几丁质酶与致病力之间的相关性;对发酵条件进行优化,摸索绿僵菌的产酶最佳条件,制备粗酶制剂;将其与微生物农药Bt进行配伍,观察协同作用效果。主要研究结果如下:
1﹑观察了10株绿僵菌对微小牛蜱饥饿幼虫的致病性作用,通过生物测定,得知Ma002菌株对微小牛蜱饥饿幼虫致病力最高,致死率达到82.1%。半数致死时间最短的是Ma002的LT_(50)为4.83 d;所以将Ma002作为所选菌株。测定了10株绿僵菌的蛋白酶和几丁质酶的活性,用生物统计学的方法分析了其与菌株对蜱的毒力之间的关系,相关系数r_1=0.8183,r_2=0.6828。结果表明,绿僵菌对微小牛蜱饥饿幼虫的毒力与其胞外酶之间呈现正向相关,并发现几丁质酶与菌株毒力之间的相关性要明显高于蛋白酶(r1>r2),检验结果极显著。
2﹑应用微生物发酵的方法研究了Ma002产几丁质酶的培养特性。Ma002产几丁质酶的最佳组分为:胶体几丁质为碳源,蛋白胨为氮源,0.05% MgSO_4·7H_2O,0.001%ZnSO_4·7H_2O。优化的培养条件:装基量12%、24 h的种子液、起始pH值7.5、发酵温度26℃、搅拌速度180 r/min、产酶时程96 h。通过条件优化,产酶菌株Ma002酶活力从1.67 U/mL提高到2.63 U/mL,酶活力提高57%。
3﹑观察了几丁质酶作为一种生物添加剂与Bt农药配伍的增效作用。结果显示,随着几丁质酶溶液浓度的提高,混合药液的杀虫效果也随之提高。当在1.6×10~2 mg/kg浓度的Bt溶液中添加浓度为2 U/mL的酶时,杀虫效果最强,累计死亡率从52%提高到85.8%,表明几丁质酶可以作为一种增效剂,提高微生物农药的作用。
Ticks are ectoparasites classified in the class Acari and parasitize on the surface of animals, which are widely spread in all over of China,constricting the development of animal industry. The parasites not only cause degrading of animal production by bloodsucking, but also transmit various pathogens, including viruses, bacteria and parasites such as Theileria and Babesia. Ticks and tick borne diseases usually cause heavy economic loss in husbandry industry. Currently, tick control is predominantly by chemical therapy. However, due to the issues concerning resistance, environmental pollution and public sanitation, the wide application of this method has been limited. Therefore, more and more scientists shed their eyes on immune prophylaxis and biocontrol.As potential factors for biocontrol,entomopathogenic fungi have been widely applied in the control of insect pests in agriculture. However, its use has just started in laboratory in control of nematodes and ectoparasites.
This study is taking pathogenic strains as target, focuses on their virulence factors----extracellular protease and chitinase, and their correlation to pathogenicity. Then the individual effects of the extracellular protease and chitinase was evaluated by induction of enzyme yields using selective cultural media. The optimized conditions for chitinase production were determined and crude enzyme was prepared. And the synergy effects in compatible with the microbial pesticide Bt was studied,which provides the basis of development for new biological agents. The major achievements are as following:
1. Through study on the pathogenecity of 10 isolates of Metarhizium against unfed larvae of B. microplus and bioassay, Ma002 strain was assessed to be the highest in pathogenecity with lethality of 82.1% and the shortest LT50 of 4.83d.The fermentation of 10 Metarhizium isolates protease and chitinase, the virulence of the strain with different yields of these enzymes was assessed by biometrics.The coefficient correlation of r1,r2 and r0.05 were 0.8183,0.6828,respectively.The results showed that there was a significant correlation, in which the virulence of the strain with high yield of chitinase is significantly higher than that of the protease (r1> r_2).
2. The characteristic of Ma002 for producing chitinase was studied by fermentation. The method showed optimal fermentation conditions was that colloid chitin was used as carbon source and peptone as nitrogen source; the inoculating periods was 24 h.The optimized cultural conditions were in flask with 12% volume medium of pH7.5-8.0 at 26℃, culturing for 96 h on 180 r/min shaker. Under these conditions, the chitinase activity increased 57% comparised to the original basal medium from 1.67 U/mL to 2.63 U/mL.
3. The effection of chitinase as biological additive was observed.By combination with Bt pesticides under a certain percentage, its synergistic effect was observed. With the increase of the concentration of chitinase, the insecticidal effect also increased. Adding a final concentration of 2 U/mLchitinase to 1.6×102 mg/kg Bt solution the cumulative mortality from 52% to 85.8% and the insecticidal effect could increase by 62.2%, indicating that chitinase could be a synergist to enhance microbial pesticides effect of significantly.
致病性病原真菌作为一种很有潜力的生物控制因子,已在农业害虫生物防治领域得到广泛地应用,而在兽医线虫和外寄生虫病防治的研究才刚刚起步。本文以对蜱具有高致病力的菌株为研究对象,研究其毒力因子——胞外蛋白酶和几丁质酶与致病力之间的相关性;对发酵条件进行优化,摸索绿僵菌的产酶最佳条件,制备粗酶制剂;将其与微生物农药Bt进行配伍,观察协同作用效果。主要研究结果如下:
1﹑观察了10株绿僵菌对微小牛蜱饥饿幼虫的致病性作用,通过生物测定,得知Ma002菌株对微小牛蜱饥饿幼虫致病力最高,致死率达到82.1%。半数致死时间最短的是Ma002的LT_(50)为4.83 d;所以将Ma002作为所选菌株。测定了10株绿僵菌的蛋白酶和几丁质酶的活性,用生物统计学的方法分析了其与菌株对蜱的毒力之间的关系,相关系数r_1=0.8183,r_2=0.6828。结果表明,绿僵菌对微小牛蜱饥饿幼虫的毒力与其胞外酶之间呈现正向相关,并发现几丁质酶与菌株毒力之间的相关性要明显高于蛋白酶(r1>r2),检验结果极显著。
2﹑应用微生物发酵的方法研究了Ma002产几丁质酶的培养特性。Ma002产几丁质酶的最佳组分为:胶体几丁质为碳源,蛋白胨为氮源,0.05% MgSO_4·7H_2O,0.001%ZnSO_4·7H_2O。优化的培养条件:装基量12%、24 h的种子液、起始pH值7.5、发酵温度26℃、搅拌速度180 r/min、产酶时程96 h。通过条件优化,产酶菌株Ma002酶活力从1.67 U/mL提高到2.63 U/mL,酶活力提高57%。
3﹑观察了几丁质酶作为一种生物添加剂与Bt农药配伍的增效作用。结果显示,随着几丁质酶溶液浓度的提高,混合药液的杀虫效果也随之提高。当在1.6×10~2 mg/kg浓度的Bt溶液中添加浓度为2 U/mL的酶时,杀虫效果最强,累计死亡率从52%提高到85.8%,表明几丁质酶可以作为一种增效剂,提高微生物农药的作用。
Ticks are ectoparasites classified in the class Acari and parasitize on the surface of animals, which are widely spread in all over of China,constricting the development of animal industry. The parasites not only cause degrading of animal production by bloodsucking, but also transmit various pathogens, including viruses, bacteria and parasites such as Theileria and Babesia. Ticks and tick borne diseases usually cause heavy economic loss in husbandry industry. Currently, tick control is predominantly by chemical therapy. However, due to the issues concerning resistance, environmental pollution and public sanitation, the wide application of this method has been limited. Therefore, more and more scientists shed their eyes on immune prophylaxis and biocontrol.As potential factors for biocontrol,entomopathogenic fungi have been widely applied in the control of insect pests in agriculture. However, its use has just started in laboratory in control of nematodes and ectoparasites.
This study is taking pathogenic strains as target, focuses on their virulence factors----extracellular protease and chitinase, and their correlation to pathogenicity. Then the individual effects of the extracellular protease and chitinase was evaluated by induction of enzyme yields using selective cultural media. The optimized conditions for chitinase production were determined and crude enzyme was prepared. And the synergy effects in compatible with the microbial pesticide Bt was studied,which provides the basis of development for new biological agents. The major achievements are as following:
1. Through study on the pathogenecity of 10 isolates of Metarhizium against unfed larvae of B. microplus and bioassay, Ma002 strain was assessed to be the highest in pathogenecity with lethality of 82.1% and the shortest LT50 of 4.83d.The fermentation of 10 Metarhizium isolates protease and chitinase, the virulence of the strain with different yields of these enzymes was assessed by biometrics.The coefficient correlation of r1,r2 and r0.05 were 0.8183,0.6828,respectively.The results showed that there was a significant correlation, in which the virulence of the strain with high yield of chitinase is significantly higher than that of the protease (r1> r_2).
2. The characteristic of Ma002 for producing chitinase was studied by fermentation. The method showed optimal fermentation conditions was that colloid chitin was used as carbon source and peptone as nitrogen source; the inoculating periods was 24 h.The optimized cultural conditions were in flask with 12% volume medium of pH7.5-8.0 at 26℃, culturing for 96 h on 180 r/min shaker. Under these conditions, the chitinase activity increased 57% comparised to the original basal medium from 1.67 U/mL to 2.63 U/mL.
3. The effection of chitinase as biological additive was observed.By combination with Bt pesticides under a certain percentage, its synergistic effect was observed. With the increase of the concentration of chitinase, the insecticidal effect also increased. Adding a final concentration of 2 U/mLchitinase to 1.6×102 mg/kg Bt solution the cumulative mortality from 52% to 85.8% and the insecticidal effect could increase by 62.2%, indicating that chitinase could be a synergist to enhance microbial pesticides effect of significantly.
引文
[1]高志华,刘敬泽.蜱类防治研究进展[J].寄生虫与医学昆虫学报,2003:(40)251-256
[2] Kapaa,G-P.and S.Hassan.2000.Entoma genous fungi as promising biopesticides for tick contro1[J]. Experimental and Applied Acarology,24:913-926
[3]周金林.蜱的功能分子研究及其应用前景[J].动物医学进展,2004: 5(1);53-56
[4]林华峰.虫生真菌研究进展[J].安徽农业大学学报,1998:25(3):251-254
[5] Sébastien Massart, Haissam M. Jijakli.2007.Use of molecular techniques to elucidate the mechanisms of action of fungal biocontrol agents [J]. Journal of Microbiological Methods,69 (2):229-241
[6]胡锦江,球孢白僵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
[7] Robert W. Sutherst The vulnerability of animal and human health to parasites under global change[J]. International Journal for Parasitology, 31(9),2001:933-948
[8] Kaaya, G.P., Hassan, S., 2000. Entomogenous fungi as promising bio-pesticides for tick control[J]. Exp. Appl. Acarol. 24, 913-926
[9] Brooks A J, Wall R. Infection of Psoroptesmites with the fungus Metarhizium anisopliae[J]. Exp. Appl. Acarol,2001, (25): 869-880
[10] Onofre, S.B., Miniuk, C.M., de Barros, N.M., Azevedo, J.L., 2001. Pathogenicity of four strains of entomopathogenic fungi against the bovine tick Boophilus microplus.Am. [J]. Vet. Res. 62, 1478–1480
[11] Lopes,R. B,Alves S B,Padulla L F,et al.Efficiency of Beauveria bassinana and Meatrhizium anisopliae formulations on Amblyomma cajennense namphae[J]. Rev Bras Parasitol Vet, 2007,16(1):27-31
[12] Samish, M., Ginsberg, H., Glazer, I., 2004. Biological control of tick.[J] Parasitology 129,s389-s403
[13] Scholte, E.J., Knols, B.G.J., Takken, W., 2006. Infection of the malaria mosquito Anopheles gambiae with the entomopathogenic fungus Metarhizium anisopliae reduces blood feeding and fecundity. [J]. Invertbr. Pathol. 91, 43-49
[14] Dana Ment, Galina Gindin, Victoria Soroker,et al.Metarhizium anisopliae conidial responses to lipids from tick cuticle and tick mammalian host surface[J]. Journal of Invertebrate Pathology,2010,103 (2):132-139
[15] Kaaya, G.P., 2000. Laboratory and field evaluation of entomogenous fungi for tick control. Ann. NY Acad. Sci. 916, 559-564
[16] Sitch, J.C., Jackson, C.W., 1997. Pre-penetration events affecting host specificity of Verticillium lecanii. Mycol. Res. 101, 535–541
[17] Yoder, J.A., Theriot, G.C., Rivers, D.B., 1996. Venom from Nasonia vitripennis alters waterloss from the ?esh ?y, Sarcophaga bullata. Entomol. Exp. Appl. 81,235-238
[18] Becker, C.M., Burr, T.J., 1994. Discontinuous wetting and survival of conidia of Venturia inaequalis on apple leaves. Phytopathology 84,372-378
[19] Elliot, S.L., Blanford, S., Thomas, M.B., 2002. Hostepathogen interactions in a varying environment: temperature, behavioural fever and fitness. Proc. Biol. Sci. 269, 1599-1607
[20] Polar, P., Moore, D., Kairo, M.T.K., Ramsubhag, A., 2008. Topically applied myco-acaricides for the control of cattle ticks: overcoming the challenges. Exp. Appl.Acarol.46,119-148
[21] Mitchell, T.G., 2002. Fungal pathogens of humans. In: Encyclopedia of Life Sciences.John Wiley & Sons, Ltd., Chichester, pp. 1-15
[22] Dillon, R.J., Charnley, A.K., 1990. Initiation of germination in conidia of the entomopathogenic fungus, Metarhizium anisopliae. Mycol. Res. 94,299–304
[23] Jarrold, S.L., Moore, D., Potter, U., Charnley, A.K., 2007. The contribution of surface waxes to pre-penetration growth of an entomopathogenic fungus on host cuticle. Mycol. Res. 111, 240–249
[24] Brooks, A., Wall, R., 2005. Horizontal transmission of fungal infection by Metarhizium anisopliae in parasitic Psoroptes mites (Acari: Psoroptidae). Biol.Control 34, 58–65
[25]邓国藩.中国经济昆虫志(第十五册,蜱螨目蜱总科)[M].北京:科学出版社,1978
[26] Bagga,S.,Hu, G., Screen, S.E., St Leger, R.J., 2004. Reconstructing the diversity of subtilisins in the pathogenic fungus Metarhizium anisopliae. Gene 324,159-169
[27] Arruda, W., Lubeck, I., Schrank, A., Vainstein, M.H., 2005. Morphological alterations of Metarhizium anisopliae during penetration of Boophilus microplus ticks. Exp. Appl. Acarol. 37, 231-244
[28] Chen, H.C., Chou, C.K., Sun, C.M., Yeh, S.F., 1997. Suppressive effects of destruxin B on hepatitis B virus surface antigene gene expression in human hepatoma cells. Antiviral Res. 34,137-144
[29] Garcia,M.V., Monteiro, A.C., Szabo,M.J.P., Prette, N., Bechara, G.H., 2005. Mechanism of infection and colonization of Rhipicephalus sanguineus eggs by Metarhizium anisopliae as revealed by scanning electron microscopy and histopathology.Braz. J. Microbiol. 36, 368-372
[30] Fang, W., Pei, Y., Bidochka, M.J., 2007. A regulator of a G protein signalling(RGS) gene, cag8, from the insect-pathogenic fungus Metarhizium anisopliae is involved in conidiation, virulence and hydrophobin synthesis. Microbiology 153, 1017-1025
[31] Jarrold, S.L., Moore, D., Potter, U., Charnley, A.K., 2007. The contribution of surface waxes to pre-penetration growth of an entomopathogenic fungus on host cuticle. Mycol. Res. 111, 240-249
[32] Silva, W.O.B., Mitidieri, S., Schrank, A., Vainstein, M.H., 2005. Production and extraction of an extracellular lipase from the entomopathogenic fungus Metarhizium anisopliae. Proc. Biochem. 40, 321-326
[33] Wang, C., St Leger, R.J., 2007. The MAD1 adhesin of Metarhizium anisopliae links adhesionwith blastospore production and virulence to insects,and the MAD2 adhesin enable attachment to plants. Eukaryot. Cell 6,808-816
[34] Frazzon, A.P., Vaz Junior, I., Masuda, A., Schrank, A., Vainstein, M.H., 2000 In vitro assessment of Metarhizium anisopliae isolates to control thecattle tick Boophilus microplus. Vet. Parasitol. 94, 117-125
[35] Barreto, C.C., Staats, C.C., Schrank, A., Vainstein, M.H., 2004. Distribution of chitinases in the entomopathogen Metarhizium anisopliae andeffect of N-acetylglucosamine in protein secretion. Curr. Microbiol. 48,102-107
[36] Da Silva, M.V., Santi, L., Staats, C.C., Costa, A.M., Colodel, E.M.,Driemeier, D., Vainstein, M.H., Schrank, A., 2005. Cuticle-induced endo/exoacting chitinase CHIT30 from Metarhizium anisopliae is encoded by an ortholog of the chi3 gene. Res. Microbiol. 156,382-392
[37] Arruda, W., Lubeck, I., Schrank, A., Vainstein, M.H., 2005. Morphological alterations of Metarhizium anisopliae during penetration of Boophilus microplus ticks. Exp. Appl. Acarol. 37, 231-244
[38] Lubeck, I., Arruda,W., Souza, B.K., Stanis?uaski, F., Carlini, C.R., Schrank, A.,Vainstein, M.H., 2008. Evaluation of Metarhizium anisopliae strains as potential biocontrol agents of the tick Rhipicephalus (Boophilus) microplus and the cotton stainer Dysdercus peruvianus. Fungal Ecol.1,78-88
[39] Boldo, J.T., Junges, A., Amaral, K.B., Staats, C.C., Vainstein,M.H., Schrank, A.,2009. Endochitinase CHI2 of the biocontrol fungus Metarhizium anisopliae affects its virulence toward the cotton stainer bug Dysdercus peruvianus. Curr. Genet.55,551-560
[40] Alonso-D?az, M.A., Garcia, L., Galindo-Velasco, E., Lezama-Gutierrez, R.,Angel-Sahagun, C.A., Rodr?guez-Vivas, R.I., Fragoso-Sanchez, H., 2007.Evaluation of Metarhizium anisopliae (Hyphomycetes) for the control of Boophilus microplus (Acari:Ixodidae) on naturally infested cattle in the Mexican tropics. Vet. Parasitol. 147, 336-340.
[41] Silva, W.O.B., Santi, L., Schrank, A., Vainstein, M.H., 2009. Metarhizium anisopliae lipolytic activity plays a pivotal role in Rhipicephalus (Boophilus) microplus infection. Mycol. Res. 2009.08.003
[42] Bischoff, J.F., Rehner, S.A., Humber, R.A., 2009. A multilocus phylogeny of the Metarhizium anisopliae lineage. Mycologia 101, 512-530
[43] St.Leger,R.J.,Charnley,A.K., Cooper, R.M., 1987. Characterization of cuticle-degrading proteases produced by the entomopathogen Metarhizium anisopliae. Arch. Biochem. Biophys. 253, 221-232
[44] St. Leger, R.J., Joshi, L., Bidochka, M.J., Rizzo, N.W., Roberts, D.W., 1996a.Characterization and ultrastructural localization of chitinases from Metarhizium anisopliae, Metarhizium ?avoviride, and Beauveria bassiana during fungal invasion of host (Manduca sexta) cuticle. Appl.Environ. Microbiol. 62, 907-912
[45] Wang, C., Typas, M.A., Butt, T.M., 2002. Detection and characterisation of pr1 virulent gene deficiencies in the insect pathogenic fungus Metarhizium anisopliae. FEMS Microbiol. Lett. 213, 251-255
[46] Small, C.L., Bidochka, M.J., 2005. Up-regulation of Prl, a subtilisin-like protease, during conidiation in the insect pathogen Metarhizium anisopliae. Mycol. Res. 109, 307-313
[47] Gillepsie JP, Bateman R, Charnley AK, 1998. Role of cuticle-de-grading proteases in the virulence of Metarhizium spp. for the desert locust, Schistocerca gregaria. Journal of Invertebrate Pathology,71: 128-137
[48] Michael J, Bidochka, Geogre G Knaehatour et al. Regulation of extraeellular protease in the enromopazhogenie fungus B.bassiana[J]. Experi Myeol, 1988, 12:161-168
[49]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
[50]彭国雄,张永军,杨星勇,等.球孢白僵菌不同世代菌株胞外蛋白酶与毒力的关系[J].中国生物防治,2000,16(2):61-64
[51] Joshi L, St Leger RJ, Bidochka MJ. Cloning of a cuticle degrading protease from the entomopathogenic fungus, Beauveria bassiana[J]. FEMS Microbiol Lett. 1995, 125(2-3): 211-217
[52]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
[53]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
[54] Butt, T.M., Ibrahim, L., Ball, B.V., Clark, S.J., 1994. Pathogenicity of the entomogenous, hyphomycete fungi Metarhizium anisopliae and Beauveria bassiana against crucifer pests and the honey bee. Biocontrol Sci. Technol.4,207-214
[55] Robert A, Messing-Al-Aidros K, 1985. Acid production by Metarhizium anisopliae: effects on virulence against mosquitoes and on detection of in vitro amylase, protease, and lipase activity. Journal of Invertebrate Pathology 45: 9-15
[56] Kunert, J., 2000. Physiology of keratinophilic fungi. In: Kushwaha, R.K.S.,Guarro, J.(Eds.), Biology of Dermatophytes and Other Keratinophilic Fungi. Revista Iberoamericana de Micología, Bilbao, pp. 77-85
[57] Magalh?es, B.P., Faria, M., Tigano, M.S., 1997. Characterization and virulence of a Brazilian isolate of Metarhizium ?avoviride Gams and Rozsypal (Hyphomycetes). Mem. Entomol. Soc.Can. 171, 313-321
[58] Staats, C.C., Junges, A., Fitarelli, M., Furlaneto, M.C., Vainstein, M.H.,Schrank, A., 2007. Gene inactivation mediated by Agrobacterium tumefaciens in the filamentous fungi Metarhizium anisopliae. Appl.Microbiol. Biotechnol. 76, 945-950
[59] Pedras, M.S.C., Zaharia, L.I., Ward, D.E., 2002. The destruxins: synthesis,biosynthesis, biotransformation, and biological activity. Phytochem-istry 59, 579-596
[60] St. Leger, R.J., Cooper, R.M., Charnley, A.K., 1991. Characterization of chitinase and chitobiase produced by the entomopathogenic fungus Metarhizium anisopliae. J. Invertebr. Pathol. 58, 415-426
[61] Pinto, A.S., Barreto, C.C., Schrank, A., Ulhoa, C.J., Vainstein, M.H., 1997. Purification and characterization of an extracellular chitinase from the entomopathogen Metarhizium anisopliae. Can. J. Microbiol. 43,322-327
[62] Kang, S.C., Park, S., Lee, D.G., 1999. Purification and characterization of a novel chitinase from the entomopathogenic fungus, Metarhizium anisopliae. J. Invertebr. Pathol. 73, 267-271
[63] Santi, L., Silva, W.O.B., Pinto, A.F., Schrank, A., Vainstein, M.H., 2009.Differential immunoproteomics enables identification of Metarhizium anisopliae proteins related to Rhipicephalus microplus infection. Res.Microbiol.2009.09.012
[64] Bogo, M.R., Rota, C.A., Pinto Jr., H., Ocampos, M., Correa, C.T., Vainstein, M.H., Schrank, A., 1998. A chitinase encoding gene (chit1 gene) from the entomopathogenic Metarhizium anisopliae: isolation and characterization of genomic and full-length cDNA. Curr. Microbiol. 37,221-225
[65] Baratto, C.M., Silva, M.V., Santi, L., Passaglia, L.M.P., Schrank, I.S.,Vainstein, M.H., Schrank, A., 2003. Expression and characterization of the 42 kDa chitinase from the biocontrol fungus Metarhizium anisopliae in Escherichia coli. Can. J. Microbiol. 49, 723-726
[66] Baratto, C.M., Dutra, V., Boldo, J.T., Leiria, L.B., Vainstein, M.H., Schrank, A.,2006. Isolation, characterization, and transcriptional analysis of the chitinase chi2 gene (DQ011663) from the biocontrol fungus Metarhizium anisopliae var. anisopliae. Curr. Microbiol. 53, 217-221
[67] Freimoser, F.M., Screen, S., Bagga, S., Hu, G., St Leger, R.J., 2003. Expressed sequence tag (EST) analysis of two subspecies of Metarhizium anisopliae reveals a plethora of secreted proteins with potential activity in insect hosts. Microbiology 49, 239-247
[68] Bittencourt, V.R.E.P., Mascarenhas, A.G., Menezes, G.C.R., Monteiro, S.G., 2000. In vitro action of the fungus Metarhizium anisopliae (Metschnikoff, 1879) Sorokin,1883 and Beauveria bassiana (Balsamo) Vuillemin,1912 on eggs of the tick Anocentor nitens (Neummann, 1897) (Acari: Ixodidae). Braz. J. Vet. Med. 22,248-251
[69] Nakazato, L., Dutra, V., Broetto, L., Staats, C.C., Vainstein, M.H., Schrank, A.,2006. Development of an expression vector for Metarhizium anisopliae based on the tef-1alpha homologous promoter. Appl. Microbiol. Biotechnol. 72, 521-528
[70] Fang W G, Leng B, Xiao Y H, et al.Cloning of Beauveria bassiana chitinase gene Bbchit l and its application to improve fungal strain virolence[J]. Appl. Environ. Microbiol. 2005, 71(1):363-370
[71]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
[72] Sohail S. Qazi, George G. Khachatourians. Addition of exogenous carbon and nitrogen sources to aphid exuviae modulates synthesis of proteases and chitinase by germinating conidia of Beauveria bassiana[J]. Archives of Microbiol, 2008, 189:589-596
[73]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
[74] Zhao, H., Charnley, A.K., Wang, Z., Yin, Y., Li, Z., Li, Y., Cao, Y., Peng, G.,Xia, Y., 2006. Identification of an extracellular acid trehalase and its gene involved in fungal pathogenesis of Metarizium anisopliae.J.Biochem. 140, 319-327
[75] Zhang, C., Xia, Y., 2009. Identification of genes differentially expressed in vivo by Metarhizium anisopliae in the hemolymph of Locusta migratoria using suppression-subtractive hybridization. Curr. Genet. 55, 399-407
[76] Hu, Q.B., Ren, S.X., Wu, J.H., Chang, J.M., Musa, P.D., 2006. Investigation of destruxin A and B from 80 Metarhizium strains in China, and the optimization of cultural conditions for the strain MaQ10. Toxicon 48,491-498
[77] Cavelier, F., Verducci, J., Andre, F., Haraux, F., Sigalat, C., Traris, M., Vey, A.,1998. Natural cyclopeptides as leads for novel pesticides: tentoxin and destruxins. Pestic. Sci. 52, 81-89
[78] Thomsen, L., Eilenberg, J., 2000. Timeeconcentration mortality of Pieris brassicae (Lepidoptera: Pieridae) and Agrotis segetum (Lepidoptera:Noctuidae) larvae from different destruxins. Environ. Entomol. 5,1041-1047
[79] Saharan, G.S., Naresh, M., Sangwan, M.S., 2003. Nature and mechanism of resistance to Alternaria blight in rapeseedemustard system. Ann. Rev.Plant Pathol. 2, 85-128
[80] Kershaw, M.J., Moorhouse, E.R., Bateman, R., Reynolds, S.E., Charnley, A.K.,1999. The role of destruxins in the pathogenicity of Metarhizium anisopliae for three species of insect. J. Invertebr. Pathol. 74, 213-223
[81] Sree, K.S., Padmaja, V., Murthy, Y.L., 2008. Insecticidal activity of destruxin, a mycotoxin from Metarhizium anisopliae (Hypocreales),against Spodoptera litura (Lepidoptera: Noctuidae) larval stages. PestManag. Sci. 64, 119-125
[82] Faria, M.R., Wraight, S.P., 2007. Mycoinsecticides and mycoacaricides:a comprehensive list with worldwide coverage and international classification of formulation types. Biol. Control 43, 237-256
[83] Milner, R.J.,2000. Current status of Metarhizium as a mycoinsecticide in Australia. Biocontrol News Inform. 21, 47-50
[84] Smirnoff W A,et al.Determination of the chitinolytic activity of nine sub-species of Bacillus thurigiensis. Journal of Invertebrate Pathology,1977,30:265-266
[85] Ding X,Gopala K B,johnson L.B.et al. Insect resistance of transgenic tobacco expression an insect chitinase gene. Transgen Res,1998,7:77-84
[86]徐建敏,余健秀,庞义.添加剂Bt-ICP杀斜纹夜蛾效果的影响.昆虫天敌,1998,20 (2):49-55
[87] Shapiro M and Preisler H K,Roberlson J L.Enhancement of haculovirus activity on gypsy moth by chitinase. J. Econ Entomol,1987,80:1113-1116
[88]徐红革,彭辉银,刘家欣. WHS3菌株产几丁质酶对棉铃虫HaSXPV的增效作用.中国病毒学,2002,17(3):239-242
[89] St.Leger, R.J., May, B., Allee, L.L., Frank, D.C., Staples, R.C., Roberts, D.W., 1992. Genetic differences in allozymes and in formation of infection structures among isolates of the entomopathogenic fungus Metarhizium anisopliae. J. Invertebr.Pathol. 60, 89-101
[90] Sayed A. Elmarakby, Farouk S. El-Feraly, Hala N. Elsohly, Edward M. Croom, Charles D. Hufford Microbiological transformations of artemisinic acid Phytochemistry, Volume 27, Issue 10, 1988, Pages 3089-3091
[91] St. Leger, R.J., Cooper, R.M., Charnley, A.K., 1991. Characterization of chitinase and chitobiase produced by the entomopathogenic fungus Metarhizium anisopliae. J. Invertebr. Pathol. 58, 415-426
[92] Gupta S C, LeathersTD, El-Sayed G N, Ignoff C M. Relationship among enzyme activities and virulence parameters in Beauveria bassianainfections of Galleri amellonella and Richoplusiani[J]. Invertebr Pathol, 1994, 64: 13-17
[93] Hajek AE, Leger St. Interactions between fungal pathogens and insect hosts. Annu Rev Entomol. 1994,39:293-322
[94] Sabatina G A, Kemp D H, Hughes S. Test to determine LC50 and discriminating dose for macrocycline lactones aginst the cattle tick Boophilius microplus[J]. Veterinary Parasitology, 2001,95:53-62
[95]唐启义.通用统计软件DPS研制与应用[J].科学中国人, 2004, (09)
[96]宋晓鸥.半数剂量的计算,李寿祺主编.卫生毒理学原理和方法.四川省科学技术出版社,1987:387
[97] Lowry O H, Rosbrough N J, Farr A L, Randdall R J. Protein measurement with folin phenol reagent. J. Biol. Chem.,1995,193:265
[98]刘智辉,陈守文,郭志红,等.球孢白僵菌胞外蛋白酶和几丁质酶活性与对亚洲玉米螟毒力的相关性分析[J ] .华中农业大学学报, 2005 , 24 (4) : 364 - 3681
[99] Runey,D.J.Probitanalysis,3rd ed.Cambridge:Cambrdge University Press 1980.1-333.
[100]聂明,魏新林,万佳蓉,等.土壤中选育产壳聚糖酶菌株的研究[J].生物技术,2005,15(5): 16-18
[101]王玉菊,祁红英,郭坚华.植物土传病害的微生物防治研究进展[J]世界农业, 1995,(01)
[102]谢晶.植物病原菌拮抗微生物的筛选、鉴定及拮抗机理研究[D].四川大学, 2004
[103] YOL-JIN-JEON, SHANIDI F, KIM SE-KWON. Preparation of chitin and chitosan chitoolingosacchrides and their applications in physiological functional foods[ J]. Food Rev Internat, 2000, 16 (2): 159-176,179
[104]姚剑,黄大庆.球孢白僵菌脂肪酸、酯酶、脂肪酶及其与毒力的关系[J].中国生物防治, 2005 , 21 (3) : 167 -171
[105] Jung WJ,An KN,Jin YL, et al. Biological control of Damping-off caused by Rhizoctonia olani using chitinase producing Paenibacillus illinoisensis KJA-424.[J]. Soil Biol.Biochem, 2003(35):1261-1264
[106]陈三凤,李季伦.几丁质酶研究历史和发展前景[J].微生物学通报.1993.20(3):156-160
[107]顾金保.昆虫微生物杀虫剂的研究进展[J].中国寄生虫病防治杂志.2005.18(6):468-470
[108] Fenice M, Giambattista DiR, Selbman L,et al. Production of N-acety-D-glucosamine and chitinolytic enzyme by a strain of Verticillum cfr.Lecanii(A3) cultivated in bench-top Fermentor [A].Advance in Chitin Science(VolumeII)[C]. Jacques Andre Publisher, 1997: 145-150
[109] Tronsmo A, Hjeljord L, Klemsdal S S, et al.Chitinolytic enzymes from the biocontrol agent Trichoderm a harrzianum [A]. In: Chitin Enzymol Proc Int Symp 2nd.[C]. Grottammare. 1996:235-244
[110]蒋红彬,张瀛,蒋千里,等.几丁质酶的研究概况[J].山东科学,2000(13):41-45
[111] Rodriguez Kabana R,Godoy G,Morga-Jones G,Shelby RA.The determination of soil chitinase activity:Conditions for assay and ecological studies[J].Plant Soil,1983(75): 95-106
[112] Joshi S,Kozlowski M,Richens S,Comberbach DM.Chitinase and chitobiase production during fermentation of genetically improved Serratia liquefaciens[J]. Enzyme Microb Technol,1989(11): 289-296
[113] Leger R,Joshi L,Roberts D.Ambient pH is a major determinant in the expression of cuticle degrading enzymes and hydrophobin by [J].Appl Enviro Microbiol,1998,64(2):709-713
[114] WU Mian-bin,XIA Li-ming,CEN Pei-lin. Random attacking model for enzymatic hydrolysis of chitosan [J]. Chem Eng of Chinese Univ,2001,15(6):552-556
[115]周兆斓,朱祯.植物抗虫基因工程研究进展.生物工程进展. 1994,14(4):18-24
[116]张继红,王深柱,钦俊德.苏云金芽抱杆菌6一内毒素的杀虫机理及其增效途径.[J].昆虫学报.1998,41(3):323一332
[117] Kramer K J,Muthukrishnan S. Insect Chintinases: molecular biology and potential use as biopesticides[J]. Insect biochem Molec Biol, 1998,27: 887-900
[118]孙红武,罗建勋,欧阳五庆,殷宏.七种药物对微小牛蜱的半数致死浓度测定[J].中国兽医科技,2004, (02)
[2] Kapaa,G-P.and S.Hassan.2000.Entoma genous fungi as promising biopesticides for tick contro1[J]. Experimental and Applied Acarology,24:913-926
[3]周金林.蜱的功能分子研究及其应用前景[J].动物医学进展,2004: 5(1);53-56
[4]林华峰.虫生真菌研究进展[J].安徽农业大学学报,1998:25(3):251-254
[5] Sébastien Massart, Haissam M. Jijakli.2007.Use of molecular techniques to elucidate the mechanisms of action of fungal biocontrol agents [J]. Journal of Microbiological Methods,69 (2):229-241
[6]胡锦江,球孢白僵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
[7] Robert W. Sutherst The vulnerability of animal and human health to parasites under global change[J]. International Journal for Parasitology, 31(9),2001:933-948
[8] Kaaya, G.P., Hassan, S., 2000. Entomogenous fungi as promising bio-pesticides for tick control[J]. Exp. Appl. Acarol. 24, 913-926
[9] Brooks A J, Wall R. Infection of Psoroptesmites with the fungus Metarhizium anisopliae[J]. Exp. Appl. Acarol,2001, (25): 869-880
[10] Onofre, S.B., Miniuk, C.M., de Barros, N.M., Azevedo, J.L., 2001. Pathogenicity of four strains of entomopathogenic fungi against the bovine tick Boophilus microplus.Am. [J]. Vet. Res. 62, 1478–1480
[11] Lopes,R. B,Alves S B,Padulla L F,et al.Efficiency of Beauveria bassinana and Meatrhizium anisopliae formulations on Amblyomma cajennense namphae[J]. Rev Bras Parasitol Vet, 2007,16(1):27-31
[12] Samish, M., Ginsberg, H., Glazer, I., 2004. Biological control of tick.[J] Parasitology 129,s389-s403
[13] Scholte, E.J., Knols, B.G.J., Takken, W., 2006. Infection of the malaria mosquito Anopheles gambiae with the entomopathogenic fungus Metarhizium anisopliae reduces blood feeding and fecundity. [J]. Invertbr. Pathol. 91, 43-49
[14] Dana Ment, Galina Gindin, Victoria Soroker,et al.Metarhizium anisopliae conidial responses to lipids from tick cuticle and tick mammalian host surface[J]. Journal of Invertebrate Pathology,2010,103 (2):132-139
[15] Kaaya, G.P., 2000. Laboratory and field evaluation of entomogenous fungi for tick control. Ann. NY Acad. Sci. 916, 559-564
[16] Sitch, J.C., Jackson, C.W., 1997. Pre-penetration events affecting host specificity of Verticillium lecanii. Mycol. Res. 101, 535–541
[17] Yoder, J.A., Theriot, G.C., Rivers, D.B., 1996. Venom from Nasonia vitripennis alters waterloss from the ?esh ?y, Sarcophaga bullata. Entomol. Exp. Appl. 81,235-238
[18] Becker, C.M., Burr, T.J., 1994. Discontinuous wetting and survival of conidia of Venturia inaequalis on apple leaves. Phytopathology 84,372-378
[19] Elliot, S.L., Blanford, S., Thomas, M.B., 2002. Hostepathogen interactions in a varying environment: temperature, behavioural fever and fitness. Proc. Biol. Sci. 269, 1599-1607
[20] Polar, P., Moore, D., Kairo, M.T.K., Ramsubhag, A., 2008. Topically applied myco-acaricides for the control of cattle ticks: overcoming the challenges. Exp. Appl.Acarol.46,119-148
[21] Mitchell, T.G., 2002. Fungal pathogens of humans. In: Encyclopedia of Life Sciences.John Wiley & Sons, Ltd., Chichester, pp. 1-15
[22] Dillon, R.J., Charnley, A.K., 1990. Initiation of germination in conidia of the entomopathogenic fungus, Metarhizium anisopliae. Mycol. Res. 94,299–304
[23] Jarrold, S.L., Moore, D., Potter, U., Charnley, A.K., 2007. The contribution of surface waxes to pre-penetration growth of an entomopathogenic fungus on host cuticle. Mycol. Res. 111, 240–249
[24] Brooks, A., Wall, R., 2005. Horizontal transmission of fungal infection by Metarhizium anisopliae in parasitic Psoroptes mites (Acari: Psoroptidae). Biol.Control 34, 58–65
[25]邓国藩.中国经济昆虫志(第十五册,蜱螨目蜱总科)[M].北京:科学出版社,1978
[26] Bagga,S.,Hu, G., Screen, S.E., St Leger, R.J., 2004. Reconstructing the diversity of subtilisins in the pathogenic fungus Metarhizium anisopliae. Gene 324,159-169
[27] Arruda, W., Lubeck, I., Schrank, A., Vainstein, M.H., 2005. Morphological alterations of Metarhizium anisopliae during penetration of Boophilus microplus ticks. Exp. Appl. Acarol. 37, 231-244
[28] Chen, H.C., Chou, C.K., Sun, C.M., Yeh, S.F., 1997. Suppressive effects of destruxin B on hepatitis B virus surface antigene gene expression in human hepatoma cells. Antiviral Res. 34,137-144
[29] Garcia,M.V., Monteiro, A.C., Szabo,M.J.P., Prette, N., Bechara, G.H., 2005. Mechanism of infection and colonization of Rhipicephalus sanguineus eggs by Metarhizium anisopliae as revealed by scanning electron microscopy and histopathology.Braz. J. Microbiol. 36, 368-372
[30] Fang, W., Pei, Y., Bidochka, M.J., 2007. A regulator of a G protein signalling(RGS) gene, cag8, from the insect-pathogenic fungus Metarhizium anisopliae is involved in conidiation, virulence and hydrophobin synthesis. Microbiology 153, 1017-1025
[31] Jarrold, S.L., Moore, D., Potter, U., Charnley, A.K., 2007. The contribution of surface waxes to pre-penetration growth of an entomopathogenic fungus on host cuticle. Mycol. Res. 111, 240-249
[32] Silva, W.O.B., Mitidieri, S., Schrank, A., Vainstein, M.H., 2005. Production and extraction of an extracellular lipase from the entomopathogenic fungus Metarhizium anisopliae. Proc. Biochem. 40, 321-326
[33] Wang, C., St Leger, R.J., 2007. The MAD1 adhesin of Metarhizium anisopliae links adhesionwith blastospore production and virulence to insects,and the MAD2 adhesin enable attachment to plants. Eukaryot. Cell 6,808-816
[34] Frazzon, A.P., Vaz Junior, I., Masuda, A., Schrank, A., Vainstein, M.H., 2000 In vitro assessment of Metarhizium anisopliae isolates to control thecattle tick Boophilus microplus. Vet. Parasitol. 94, 117-125
[35] Barreto, C.C., Staats, C.C., Schrank, A., Vainstein, M.H., 2004. Distribution of chitinases in the entomopathogen Metarhizium anisopliae andeffect of N-acetylglucosamine in protein secretion. Curr. Microbiol. 48,102-107
[36] Da Silva, M.V., Santi, L., Staats, C.C., Costa, A.M., Colodel, E.M.,Driemeier, D., Vainstein, M.H., Schrank, A., 2005. Cuticle-induced endo/exoacting chitinase CHIT30 from Metarhizium anisopliae is encoded by an ortholog of the chi3 gene. Res. Microbiol. 156,382-392
[37] Arruda, W., Lubeck, I., Schrank, A., Vainstein, M.H., 2005. Morphological alterations of Metarhizium anisopliae during penetration of Boophilus microplus ticks. Exp. Appl. Acarol. 37, 231-244
[38] Lubeck, I., Arruda,W., Souza, B.K., Stanis?uaski, F., Carlini, C.R., Schrank, A.,Vainstein, M.H., 2008. Evaluation of Metarhizium anisopliae strains as potential biocontrol agents of the tick Rhipicephalus (Boophilus) microplus and the cotton stainer Dysdercus peruvianus. Fungal Ecol.1,78-88
[39] Boldo, J.T., Junges, A., Amaral, K.B., Staats, C.C., Vainstein,M.H., Schrank, A.,2009. Endochitinase CHI2 of the biocontrol fungus Metarhizium anisopliae affects its virulence toward the cotton stainer bug Dysdercus peruvianus. Curr. Genet.55,551-560
[40] Alonso-D?az, M.A., Garcia, L., Galindo-Velasco, E., Lezama-Gutierrez, R.,Angel-Sahagun, C.A., Rodr?guez-Vivas, R.I., Fragoso-Sanchez, H., 2007.Evaluation of Metarhizium anisopliae (Hyphomycetes) for the control of Boophilus microplus (Acari:Ixodidae) on naturally infested cattle in the Mexican tropics. Vet. Parasitol. 147, 336-340.
[41] Silva, W.O.B., Santi, L., Schrank, A., Vainstein, M.H., 2009. Metarhizium anisopliae lipolytic activity plays a pivotal role in Rhipicephalus (Boophilus) microplus infection. Mycol. Res. 2009.08.003
[42] Bischoff, J.F., Rehner, S.A., Humber, R.A., 2009. A multilocus phylogeny of the Metarhizium anisopliae lineage. Mycologia 101, 512-530
[43] St.Leger,R.J.,Charnley,A.K., Cooper, R.M., 1987. Characterization of cuticle-degrading proteases produced by the entomopathogen Metarhizium anisopliae. Arch. Biochem. Biophys. 253, 221-232
[44] St. Leger, R.J., Joshi, L., Bidochka, M.J., Rizzo, N.W., Roberts, D.W., 1996a.Characterization and ultrastructural localization of chitinases from Metarhizium anisopliae, Metarhizium ?avoviride, and Beauveria bassiana during fungal invasion of host (Manduca sexta) cuticle. Appl.Environ. Microbiol. 62, 907-912
[45] Wang, C., Typas, M.A., Butt, T.M., 2002. Detection and characterisation of pr1 virulent gene deficiencies in the insect pathogenic fungus Metarhizium anisopliae. FEMS Microbiol. Lett. 213, 251-255
[46] Small, C.L., Bidochka, M.J., 2005. Up-regulation of Prl, a subtilisin-like protease, during conidiation in the insect pathogen Metarhizium anisopliae. Mycol. Res. 109, 307-313
[47] Gillepsie JP, Bateman R, Charnley AK, 1998. Role of cuticle-de-grading proteases in the virulence of Metarhizium spp. for the desert locust, Schistocerca gregaria. Journal of Invertebrate Pathology,71: 128-137
[48] Michael J, Bidochka, Geogre G Knaehatour et al. Regulation of extraeellular protease in the enromopazhogenie fungus B.bassiana[J]. Experi Myeol, 1988, 12:161-168
[49]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
[50]彭国雄,张永军,杨星勇,等.球孢白僵菌不同世代菌株胞外蛋白酶与毒力的关系[J].中国生物防治,2000,16(2):61-64
[51] Joshi L, St Leger RJ, Bidochka MJ. Cloning of a cuticle degrading protease from the entomopathogenic fungus, Beauveria bassiana[J]. FEMS Microbiol Lett. 1995, 125(2-3): 211-217
[52]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
[53]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
[54] Butt, T.M., Ibrahim, L., Ball, B.V., Clark, S.J., 1994. Pathogenicity of the entomogenous, hyphomycete fungi Metarhizium anisopliae and Beauveria bassiana against crucifer pests and the honey bee. Biocontrol Sci. Technol.4,207-214
[55] Robert A, Messing-Al-Aidros K, 1985. Acid production by Metarhizium anisopliae: effects on virulence against mosquitoes and on detection of in vitro amylase, protease, and lipase activity. Journal of Invertebrate Pathology 45: 9-15
[56] Kunert, J., 2000. Physiology of keratinophilic fungi. In: Kushwaha, R.K.S.,Guarro, J.(Eds.), Biology of Dermatophytes and Other Keratinophilic Fungi. Revista Iberoamericana de Micología, Bilbao, pp. 77-85
[57] Magalh?es, B.P., Faria, M., Tigano, M.S., 1997. Characterization and virulence of a Brazilian isolate of Metarhizium ?avoviride Gams and Rozsypal (Hyphomycetes). Mem. Entomol. Soc.Can. 171, 313-321
[58] Staats, C.C., Junges, A., Fitarelli, M., Furlaneto, M.C., Vainstein, M.H.,Schrank, A., 2007. Gene inactivation mediated by Agrobacterium tumefaciens in the filamentous fungi Metarhizium anisopliae. Appl.Microbiol. Biotechnol. 76, 945-950
[59] Pedras, M.S.C., Zaharia, L.I., Ward, D.E., 2002. The destruxins: synthesis,biosynthesis, biotransformation, and biological activity. Phytochem-istry 59, 579-596
[60] St. Leger, R.J., Cooper, R.M., Charnley, A.K., 1991. Characterization of chitinase and chitobiase produced by the entomopathogenic fungus Metarhizium anisopliae. J. Invertebr. Pathol. 58, 415-426
[61] Pinto, A.S., Barreto, C.C., Schrank, A., Ulhoa, C.J., Vainstein, M.H., 1997. Purification and characterization of an extracellular chitinase from the entomopathogen Metarhizium anisopliae. Can. J. Microbiol. 43,322-327
[62] Kang, S.C., Park, S., Lee, D.G., 1999. Purification and characterization of a novel chitinase from the entomopathogenic fungus, Metarhizium anisopliae. J. Invertebr. Pathol. 73, 267-271
[63] Santi, L., Silva, W.O.B., Pinto, A.F., Schrank, A., Vainstein, M.H., 2009.Differential immunoproteomics enables identification of Metarhizium anisopliae proteins related to Rhipicephalus microplus infection. Res.Microbiol.2009.09.012
[64] Bogo, M.R., Rota, C.A., Pinto Jr., H., Ocampos, M., Correa, C.T., Vainstein, M.H., Schrank, A., 1998. A chitinase encoding gene (chit1 gene) from the entomopathogenic Metarhizium anisopliae: isolation and characterization of genomic and full-length cDNA. Curr. Microbiol. 37,221-225
[65] Baratto, C.M., Silva, M.V., Santi, L., Passaglia, L.M.P., Schrank, I.S.,Vainstein, M.H., Schrank, A., 2003. Expression and characterization of the 42 kDa chitinase from the biocontrol fungus Metarhizium anisopliae in Escherichia coli. Can. J. Microbiol. 49, 723-726
[66] Baratto, C.M., Dutra, V., Boldo, J.T., Leiria, L.B., Vainstein, M.H., Schrank, A.,2006. Isolation, characterization, and transcriptional analysis of the chitinase chi2 gene (DQ011663) from the biocontrol fungus Metarhizium anisopliae var. anisopliae. Curr. Microbiol. 53, 217-221
[67] Freimoser, F.M., Screen, S., Bagga, S., Hu, G., St Leger, R.J., 2003. Expressed sequence tag (EST) analysis of two subspecies of Metarhizium anisopliae reveals a plethora of secreted proteins with potential activity in insect hosts. Microbiology 49, 239-247
[68] Bittencourt, V.R.E.P., Mascarenhas, A.G., Menezes, G.C.R., Monteiro, S.G., 2000. In vitro action of the fungus Metarhizium anisopliae (Metschnikoff, 1879) Sorokin,1883 and Beauveria bassiana (Balsamo) Vuillemin,1912 on eggs of the tick Anocentor nitens (Neummann, 1897) (Acari: Ixodidae). Braz. J. Vet. Med. 22,248-251
[69] Nakazato, L., Dutra, V., Broetto, L., Staats, C.C., Vainstein, M.H., Schrank, A.,2006. Development of an expression vector for Metarhizium anisopliae based on the tef-1alpha homologous promoter. Appl. Microbiol. Biotechnol. 72, 521-528
[70] Fang W G, Leng B, Xiao Y H, et al.Cloning of Beauveria bassiana chitinase gene Bbchit l and its application to improve fungal strain virolence[J]. Appl. Environ. Microbiol. 2005, 71(1):363-370
[71]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
[72] Sohail S. Qazi, George G. Khachatourians. Addition of exogenous carbon and nitrogen sources to aphid exuviae modulates synthesis of proteases and chitinase by germinating conidia of Beauveria bassiana[J]. Archives of Microbiol, 2008, 189:589-596
[73]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
[74] Zhao, H., Charnley, A.K., Wang, Z., Yin, Y., Li, Z., Li, Y., Cao, Y., Peng, G.,Xia, Y., 2006. Identification of an extracellular acid trehalase and its gene involved in fungal pathogenesis of Metarizium anisopliae.J.Biochem. 140, 319-327
[75] Zhang, C., Xia, Y., 2009. Identification of genes differentially expressed in vivo by Metarhizium anisopliae in the hemolymph of Locusta migratoria using suppression-subtractive hybridization. Curr. Genet. 55, 399-407
[76] Hu, Q.B., Ren, S.X., Wu, J.H., Chang, J.M., Musa, P.D., 2006. Investigation of destruxin A and B from 80 Metarhizium strains in China, and the optimization of cultural conditions for the strain MaQ10. Toxicon 48,491-498
[77] Cavelier, F., Verducci, J., Andre, F., Haraux, F., Sigalat, C., Traris, M., Vey, A.,1998. Natural cyclopeptides as leads for novel pesticides: tentoxin and destruxins. Pestic. Sci. 52, 81-89
[78] Thomsen, L., Eilenberg, J., 2000. Timeeconcentration mortality of Pieris brassicae (Lepidoptera: Pieridae) and Agrotis segetum (Lepidoptera:Noctuidae) larvae from different destruxins. Environ. Entomol. 5,1041-1047
[79] Saharan, G.S., Naresh, M., Sangwan, M.S., 2003. Nature and mechanism of resistance to Alternaria blight in rapeseedemustard system. Ann. Rev.Plant Pathol. 2, 85-128
[80] Kershaw, M.J., Moorhouse, E.R., Bateman, R., Reynolds, S.E., Charnley, A.K.,1999. The role of destruxins in the pathogenicity of Metarhizium anisopliae for three species of insect. J. Invertebr. Pathol. 74, 213-223
[81] Sree, K.S., Padmaja, V., Murthy, Y.L., 2008. Insecticidal activity of destruxin, a mycotoxin from Metarhizium anisopliae (Hypocreales),against Spodoptera litura (Lepidoptera: Noctuidae) larval stages. PestManag. Sci. 64, 119-125
[82] Faria, M.R., Wraight, S.P., 2007. Mycoinsecticides and mycoacaricides:a comprehensive list with worldwide coverage and international classification of formulation types. Biol. Control 43, 237-256
[83] Milner, R.J.,2000. Current status of Metarhizium as a mycoinsecticide in Australia. Biocontrol News Inform. 21, 47-50
[84] Smirnoff W A,et al.Determination of the chitinolytic activity of nine sub-species of Bacillus thurigiensis. Journal of Invertebrate Pathology,1977,30:265-266
[85] Ding X,Gopala K B,johnson L.B.et al. Insect resistance of transgenic tobacco expression an insect chitinase gene. Transgen Res,1998,7:77-84
[86]徐建敏,余健秀,庞义.添加剂Bt-ICP杀斜纹夜蛾效果的影响.昆虫天敌,1998,20 (2):49-55
[87] Shapiro M and Preisler H K,Roberlson J L.Enhancement of haculovirus activity on gypsy moth by chitinase. J. Econ Entomol,1987,80:1113-1116
[88]徐红革,彭辉银,刘家欣. WHS3菌株产几丁质酶对棉铃虫HaSXPV的增效作用.中国病毒学,2002,17(3):239-242
[89] St.Leger, R.J., May, B., Allee, L.L., Frank, D.C., Staples, R.C., Roberts, D.W., 1992. Genetic differences in allozymes and in formation of infection structures among isolates of the entomopathogenic fungus Metarhizium anisopliae. J. Invertebr.Pathol. 60, 89-101
[90] Sayed A. Elmarakby, Farouk S. El-Feraly, Hala N. Elsohly, Edward M. Croom, Charles D. Hufford Microbiological transformations of artemisinic acid Phytochemistry, Volume 27, Issue 10, 1988, Pages 3089-3091
[91] St. Leger, R.J., Cooper, R.M., Charnley, A.K., 1991. Characterization of chitinase and chitobiase produced by the entomopathogenic fungus Metarhizium anisopliae. J. Invertebr. Pathol. 58, 415-426
[92] Gupta S C, LeathersTD, El-Sayed G N, Ignoff C M. Relationship among enzyme activities and virulence parameters in Beauveria bassianainfections of Galleri amellonella and Richoplusiani[J]. Invertebr Pathol, 1994, 64: 13-17
[93] Hajek AE, Leger St. Interactions between fungal pathogens and insect hosts. Annu Rev Entomol. 1994,39:293-322
[94] Sabatina G A, Kemp D H, Hughes S. Test to determine LC50 and discriminating dose for macrocycline lactones aginst the cattle tick Boophilius microplus[J]. Veterinary Parasitology, 2001,95:53-62
[95]唐启义.通用统计软件DPS研制与应用[J].科学中国人, 2004, (09)
[96]宋晓鸥.半数剂量的计算,李寿祺主编.卫生毒理学原理和方法.四川省科学技术出版社,1987:387
[97] Lowry O H, Rosbrough N J, Farr A L, Randdall R J. Protein measurement with folin phenol reagent. J. Biol. Chem.,1995,193:265
[98]刘智辉,陈守文,郭志红,等.球孢白僵菌胞外蛋白酶和几丁质酶活性与对亚洲玉米螟毒力的相关性分析[J ] .华中农业大学学报, 2005 , 24 (4) : 364 - 3681
[99] Runey,D.J.Probitanalysis,3rd ed.Cambridge:Cambrdge University Press 1980.1-333.
[100]聂明,魏新林,万佳蓉,等.土壤中选育产壳聚糖酶菌株的研究[J].生物技术,2005,15(5): 16-18
[101]王玉菊,祁红英,郭坚华.植物土传病害的微生物防治研究进展[J]世界农业, 1995,(01)
[102]谢晶.植物病原菌拮抗微生物的筛选、鉴定及拮抗机理研究[D].四川大学, 2004
[103] YOL-JIN-JEON, SHANIDI F, KIM SE-KWON. Preparation of chitin and chitosan chitoolingosacchrides and their applications in physiological functional foods[ J]. Food Rev Internat, 2000, 16 (2): 159-176,179
[104]姚剑,黄大庆.球孢白僵菌脂肪酸、酯酶、脂肪酶及其与毒力的关系[J].中国生物防治, 2005 , 21 (3) : 167 -171
[105] Jung WJ,An KN,Jin YL, et al. Biological control of Damping-off caused by Rhizoctonia olani using chitinase producing Paenibacillus illinoisensis KJA-424.[J]. Soil Biol.Biochem, 2003(35):1261-1264
[106]陈三凤,李季伦.几丁质酶研究历史和发展前景[J].微生物学通报.1993.20(3):156-160
[107]顾金保.昆虫微生物杀虫剂的研究进展[J].中国寄生虫病防治杂志.2005.18(6):468-470
[108] Fenice M, Giambattista DiR, Selbman L,et al. Production of N-acety-D-glucosamine and chitinolytic enzyme by a strain of Verticillum cfr.Lecanii(A3) cultivated in bench-top Fermentor [A].Advance in Chitin Science(VolumeII)[C]. Jacques Andre Publisher, 1997: 145-150
[109] Tronsmo A, Hjeljord L, Klemsdal S S, et al.Chitinolytic enzymes from the biocontrol agent Trichoderm a harrzianum [A]. In: Chitin Enzymol Proc Int Symp 2nd.[C]. Grottammare. 1996:235-244
[110]蒋红彬,张瀛,蒋千里,等.几丁质酶的研究概况[J].山东科学,2000(13):41-45
[111] Rodriguez Kabana R,Godoy G,Morga-Jones G,Shelby RA.The determination of soil chitinase activity:Conditions for assay and ecological studies[J].Plant Soil,1983(75): 95-106
[112] Joshi S,Kozlowski M,Richens S,Comberbach DM.Chitinase and chitobiase production during fermentation of genetically improved Serratia liquefaciens[J]. Enzyme Microb Technol,1989(11): 289-296
[113] Leger R,Joshi L,Roberts D.Ambient pH is a major determinant in the expression of cuticle degrading enzymes and hydrophobin by [J].Appl Enviro Microbiol,1998,64(2):709-713
[114] WU Mian-bin,XIA Li-ming,CEN Pei-lin. Random attacking model for enzymatic hydrolysis of chitosan [J]. Chem Eng of Chinese Univ,2001,15(6):552-556
[115]周兆斓,朱祯.植物抗虫基因工程研究进展.生物工程进展. 1994,14(4):18-24
[116]张继红,王深柱,钦俊德.苏云金芽抱杆菌6一内毒素的杀虫机理及其增效途径.[J].昆虫学报.1998,41(3):323一332
[117] Kramer K J,Muthukrishnan S. Insect Chintinases: molecular biology and potential use as biopesticides[J]. Insect biochem Molec Biol, 1998,27: 887-900
[118]孙红武,罗建勋,欧阳五庆,殷宏.七种药物对微小牛蜱的半数致死浓度测定[J].中国兽医科技,2004, (02)