蜜蜂球囊菌同工酶研究及聚类分析
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摘要
蜜蜂白垩病(Bee Chalkbrood Disease)是由蜜蜂球囊菌(Ascosphaera apis)寄生而引起蜜蜂幼虫死亡的真菌性传染病。对采自福建、江西、甘肃和浙江的10株蜜蜂球囊菌菌株进行了分离纯化，并且将这10个不同的菌株采用聚丙烯酰胺凝胶(或淀粉胶)电泳的方法，进行了可溶性蛋白、酯酶同工酶(EST)、苹果酸脱氢酶同工酶(MDH)、乳酸脱氢酶同工酶(LDH)、过氧化物酶同工酶(POD)、淀粉酶同工酶(AMY)和葡糖-6-磷酸脱氢酶同工酶(G-6-PDH)的研究。结果表明，各菌株间的有些同工酶酶带存在明显的差异，可溶性蛋白、EST谱带较多，MDH、LDH、POD谱带较少，且LDH、POD谱带仅在一株菌中存在，AMY的谱带不是很清晰，所有的菌株均具有相同的G-6-PDH同工酶谱带，只是浓度不一样。在所分析10株菌株中，表现出谱带的多型性。利用DPS V8.01版统计分析软件对10株菌的可溶性蛋白和其它6种同工酶进行同源性聚类分析，采用不同的距离系数和聚类方法，可得到相同的结果，10株菌可分为3个类群。结合谱带特征，筛选到一株非常特别的10号菌株。通过分析，我们发现在本文中用到的聚类方法，以可变类平均法和离差平方和法较好。同时本研究还探索了在一块胶板上同时染MDH和EST的方法。
Bee Chalkbrood Disease is a fungal infectious diseases which caused by the Ascosphaera apis and can kill the larvae. Collected from Fujian, Jiangxi, Gansu Province, 10 strains of Ascosphaera apis were isolated and purified. Meanwhile we use the methods of polyacrylamide gel (or starch gel) electrophoresis to research the 10 different strains of Soluble Protein, EST, MDH, LDH, POD, AMY and G-6-PDH. The results show that some of the strains have significantly different isozymes. Soluble protein, and EST have more bands, MDH, LDH, POD bands less, Also LDH, POD bands exist only in one strain. AMY band is not very clear. All the strains have the same bands of G-6-PDH, but not the same concentration. The 10 strains in the analysis show multi-type bands. Using DPS , a statistical analysis software of V8. 01, we conduct the homologous cluster analysis of soluble protein and other six isozymes. It show that different distance coefficient and clustering method can lead to the same results and we divided the ten strains into three group. Combined with bands, we screened a very special strain. Through analysis, we found that the clustering method used in this paper, variable types and the average square deviation are better than others. In addition, the study also explore the method that MDH and EST are dyed on the same piece of gel.
Bee Chalkbrood Disease is a fungal infectious diseases which caused by the Ascosphaera apis and can kill the larvae. Collected from Fujian, Jiangxi, Gansu Province, 10 strains of Ascosphaera apis were isolated and purified. Meanwhile we use the methods of polyacrylamide gel (or starch gel) electrophoresis to research the 10 different strains of Soluble Protein, EST, MDH, LDH, POD, AMY and G-6-PDH. The results show that some of the strains have significantly different isozymes. Soluble protein, and EST have more bands, MDH, LDH, POD bands less, Also LDH, POD bands exist only in one strain. AMY band is not very clear. All the strains have the same bands of G-6-PDH, but not the same concentration. The 10 strains in the analysis show multi-type bands. Using DPS , a statistical analysis software of V8. 01, we conduct the homologous cluster analysis of soluble protein and other six isozymes. It show that different distance coefficient and clustering method can lead to the same results and we divided the ten strains into three group. Combined with bands, we screened a very special strain. Through analysis, we found that the clustering method used in this paper, variable types and the average square deviation are better than others. In addition, the study also explore the method that MDH and EST are dyed on the same piece of gel.
引文
[1] He H, Janso JE, et al. Culicinin D, an antitumor peptaibol produced by the fungus Culicinomyces clavisporus, strain LL-121252. J Nat Prod, 2006, 69 (5): 736～777
[2] 徐伟松，李建丰，胡美英．昆虫病原真菌在害虫防治上的应用．广西农业科学，2003，6：51～52
[3] 胡奇，刘戬，闫妍．昆虫病原真菌研究进展．天津农学院学报，2004，11(4)：46～51
[4] Iskandarov US, Guzalova AG, Davranov KD. Effects of nutrient medium composition and temperature on the germination of conidia and the entomopathogenic activity of the fungi. Prikl Biokhim Mikrobiol, 2006, 42(1): 81～86
[5] Xu CP, Sinha J, et al. Optimization of physical parameters for exo-biopolymer production in submerged mycelial cultures of two entomopathogenic fungi Paecilomyces japonica and Paecilomyces tenuipes. Lett Appl Microbiol, 2006, 42(5): 501～507
[6] 张素芬．国外昆虫病原真菌研究动态．天津农林科技，1997，140(2)：40～42
[7] Cho EM, Liu L, Farmerie W, Keyhani NO. EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. Ⅰ. Evidence for stage-specific gene expression in aerial conidia, in vitro blastospores and submerged conidia. Microbiology, 2006, 152(9): 2843～2897
[8] Cho EM, Boucias D, Keyhani NO. EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. Ⅱ. Fungal cells sporulating on chitin and producing oosporein. Microbiology, 2006, 152(9): 2855～2919
[9] 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．中国生物防治，1998，14(3)：123～129
[10] Crespo R, Pedrini N, et al. Volatile organic compounds released by the entomopathogenic fungus Beauveria bassiana. Microbiol Res, 2006, 27
[11] 王成树，李增智．虫生真菌分子生物学研究进展．微生物学通报，2001，28(3)：88～92
[12] No authors. The effect of entomopathogenic fungi on the activity of detoxicating enzymes of larvae of bee pyralid Galleria mellonella L. (Lepidoptera, Pyralidee) and the role of detoxicating enzymes in the formation of the insect' resistance to entomopathogenic fungi. Izv Akad Nauk Ser Biol, 2006, (6): 712～720
[13] Pedrini N, Crespo R, Juarez MP. Biochemistry of insect epicuticle degradation by entomopathogenic fungi. Comp Biochem Physiol C Toxicol Pharmacol, 2006, 30
[14] 李建庆，张永安等．昆虫病原真菌毒素的研究进展．林业科学研究，2003，16(2)：233～239
[15] ParkNS, Lee KS, etal. Molecular cloning, expression, and characterition of the Cu, Zn superoxide dismutase (SOD1) gene from the entomopathogenic fungus Cordyceps militaris. Mycologia, 2005, 97(1): 130～138
[16] 赵继红，李建中．农用微生物杀菌剂研究进展．农药，2003，42(5)：6～8
[17] 张丽萍，张贵云．微生物农药研究进展．北京农业科学，2000，18(4)：22～24
[18] 裴炎，方卫国，张永军．昆虫病原真菌致病寄主的机制和基因工程改良．农业生物技术学报，2003，11(3)：221～226
[19] 李勇，杨慧敏．微生物农药的研究和应用进展．贵州农业科学，2003，31(2)：62～63
[20] 周传恩．我国杀虫微生物的应用研究进展及发展前景．农药，2001，40(7)：8～10
[21] Hermann Strasser, Alain Vey, Tariq M. Butt. Are There any Risks in Using Entomopathogenic Fungi for Pest Control, with Particular Reference to the Bioactive Metabolites of Metarhizlum, Tolypocladium and Beauverla species?. Biocontrol Science and Technology, 2000, 10(6):717～735
[22] Cuthbertson AG, North JP, Walters KF. Effect of temperature and host plant leaf morphology on the efficacy of two entomopathogenic biocontrol agents of Thrips palmi (Thysanoptera: Thripidae).Bull Entomol Res, 2005,95(4):321～327
[23] Lee SY, Nakajima I, et al. Cultivation of entomopathogenic fungi for the search of antibacterial compounds. Mycopathologia, 2005, 160(4):321～326
[24] 林超，孙辉等．菌寄生真菌的研究进展．菌物研究，2005，3(1)：46～52
[25] Marannino P, Santiago-Alvarez C, et al. A new bioassay method reveals pathogenicity of Metarhizium anisopliae and Beauveria bassiana against early stages of Capnodis tenebrionis (Coleoptera; Buprestidae). J Invertebr Pathol, 2006,93(3):210～213
[26] Majchrowicz, I; Poprawski, TJ. Effects in vitro of nine fungicides on growth of entomopathogenic fungi. Biocontrol Science and Technology, 1993,3(3):321～336
[27] 李多川．菌寄生真菌分子生物学研究进展．吉林农业大学学报，1998，20：37～39
[28] 邢晓科，郭顺星．菌寄生真菌与寄主相互作用的生化研究进展．菌物系统，2002，21(1)：141～146
[29] 陈大福，梁勤．蜜蜂白垩病的研究进展(连载一)．蜜蜂杂志，2001，1：3～4
[30] 李真银．中西药结合防治蜜蜂白垩病．中国兽医杂志，1994，10：37
[31] 朱亮光，刘嵋恩，何铠光．台湾蜜蜂白垩病发生之初报[J]．台湾大学植物病虫害，1984，(11)：5～7
[32] 董秉义．白垩病的诊断．中国养蜂，1992，(2)：39—40
[33] 陈淑静，康雪冬，冯峰．蜜蜂白垩病研究初报[J]．中国养蜂，1994，(6)：4～7
[34] Zaghloul OA, Mourad AK, El Kady MB et al. Assessment of losses in honey yield due to the chalkbrood disease, with reference to the determination of its economic injury levels in Egypt. Commun Agric Appl Biol Sci, 2005,70(4):703～717
[35] 郭志儒．中华人民共和国农业部第53号令公布动物病原微生物分类名录．中国兽医学报，2005，25(5)：473
[36] X. Qin, J. D. Evans, et al. Genome sequences of the honey bee pathogens Paenibacillus larvae and Ascosphaera apis Insect. Molecular Biology, 2006, 15(5):715～718
[37] 王建鼎，梁勤，苏荣．蜜蜂保护学．中国农业出版社，1997
[38] 邵瑞宜．蜜蜂白垩病病原观察及其综合防治措施．福建农林大学学报，1997，26(3)：345～348
[39] 余林生，孟祥金．蜜蜂白垩病的病原和防治技术研究．中国兽医科技，1999，29(12)：6～8
[40] Heath L A F. Chalkbrood pathogen, A review. Bee World, 1982,63(4): 130～135
[41] Gilliam M. Infectivity and survival of the chalkbrood pathogen, Ascosphaera apis in colonies of honeybee Apis mellifera. Apidologie, 1986,17:93～100
[42] Liang kuang Chu, Thomas Meien Liu, Kai kuang Ho. Growth inhibition of Ascosphaera apis by royal jelly and hydroxydecenoic acid, Reprinted from Bulletin of the Institute of Zoology. Academia sinica, 1992,31(2):73～79
[43] Lu R, Goerzen DW et al. Use of RhPD analysis for in situ identification of Ascosphaera aggregata and Ascosphaera larvis in larval cadavers of the alfalfa leafcutting bee, Megachile totundata. J Invertebr Pathol, 1996,68(1):78～83
[44] R. R. James and J.S. Skinner. PCR diagnostic methods for Ascosphaera infections in bees. Journal of Invertebrate Pathology, 2005,90:98～103
[45] 梁勤，陈大福，王建鼎．温度、相对湿度和pH对蜜蜂球囊菌孢子萌发的影响．应用生态学报，2000，11(6)：869～872
[46] James RR, Buckner JS. Lipids stimulate spore germination in the entomopathogenic ascomycete Ascosphaera aggregata. Mycopathologia, 2004, 158(3):293～302
[47] 牟方喆．蜂蜜中蜜蜂子囊球菌检测规范．吉林粮食高等专科学校学报，1999，14(4)：7～12
[48] 余林生，孟祥金等．蜜蜂白垩病发病规律与防治措施的研究．养蜂科技，1999，3：4～7
[49] 黄通明．蜜蜂白垩病的治疗．养殖天地，2002，148(6)：26
[50] Pawet Chorbinski. The development of the infection of Apismellifera larvae by Ascosphaora apis. Electronic Journal of Polish Agricultural Universities, Veterinary Medicine, 2004, 7(2)
[51] 王志，薛运波．试论蜂场生态与蜜蜂病敌害发生的关系．中国养蜂，2005，56(3)：23
[52] 时丽君．蜜蜂白垩病发病规律及防治措施．吉林畜牧兽医，2003，12：45～46
[53] 张华瑞．试述蜜蜂白垩病的流行特点与致病因素．养蜂科技，1994，4：16～17
[54] R. R. James and T. L. Pitts - Singer. Ascosphaera aggregata contamination on alfalfa leafcutting bees in a loose cell incubation system [J]. Journal of Invertebrate Pathology, 2005,89(2):176～178
[55] John. D. Vandenberg. Bioassay of the chalk brood fungus Ascosphaera aggregata on larvae of the alfalfa leafcutting bee, Megachile rotundata[J]. Journal of Invertebrate Pathology, 1992,60(2):159～163
[56] John D. Vandenberg, Becy L. Fichter, and W. P. Stephen. Spore Load of Ascosphaera Species on Emerging Adults of the Alfalfa Leafcutting Bee, Megachile rotundatat. Applied and Environmental Microbiology, 1980,39(3): 650～655
[57] 范正友．蜜蜂白垩病流行病学研究进展．中国养蜂，1996，132(1)：15～16
[58] Flores JM, Gutierrez I, Espejo R. The role of pollen in chalkbrood disease in Apis mellifera: transmission and predisposing conditions. Mycologia, 2005,97(6):1171～1176
[59] Flores JM, Spivak M, Gutierrez I. Spores of Ascosphaera apis contained in wax foundation can infect honeybee brood. Vet Microbiol, 2005, 108(1-2):141～145
[60] 牛庆生．长白山区蜜蜂白垩病及其防治[J]．吉林畜牧兽医，1997，(7)：36
[61] 李怀军．浅析呼盟地区白垩病复发的因素．蜜蜂杂志，2005，6：23～24
[62] 李真银．中西药结合防治蜜蜂白垩病．中国兽医杂志，1994，10：37
[63] J. M. Flores, I. Gutierrez and F. Puerta. Oxytetracycline as a predisposing condition for chalk brood in honey bee [J]. Veterinary Microbiology, 2004, 103(3):195～199
[64] Martha Gilliam. Identification and roles of non-pathogenic microflora associated with honey bees[J]. FEMSMicrobiology Letters, 1997,155(1):1～10
[65] 吴殿军，吴永茂等．蜜蜂检疫和二类传染病的检疫处理．广西畜牧兽医，2005，21(5)：234～235
[66] 王桂忠．蜜蜂白垩病的检疫报告．宁夏农学院学报，1997，18(2)：88～89
[67] 郑祥义，李真银．蜜蜂白垩病综合防治初探．养蜂科技，2002，4：14～15
[68] Anderson DL; Giacon H; Gibson NL. Culture, detection and thermal destruction of the chalkbrood fungus, Ascosphaera apis. Journal of Apicultural Research, 1997,36:163～168.
[69] James RR. Impact of disinfecting nesting boards on chalkbrood control in the alfalfa leafcutting bee. J Econ Entomol, 2005,98(4):1094～1100
[70] 李怀军．防治白垩病关键在消毒．蜜蜂杂志(月刊)，2005，12：28
[71] 陈大福，梁勤．蜜蜂白垩病的研究进展(连载二)．蜜蜂杂志，2001，2：10～13
[72] 王福仁．白垩病的综合防治方法．中国养蜂，1995，2：11
[73] 陈东海，历延芳等．谈长白山区蜜蜂白垩病发病率低的因素．中国养蜂，1996，5：17
[74] Tarpy DR, Seeley TD. Lower disease infections in honeybee (Apis mellifera) colonies headed by polyandrous vs monandrous queen. Naturwissenschaften, 2006, 93(4): 195～204
[75] 王志．蜜蜂种群习性与蜂病流行的关系．养蜂科技，20047，3：22～23
[76] 罗勤．抗白垩病蜂群的选育实践．蜜蜂杂志，2001，11：22～23
[77] 徐家胃．培育抗白垩病蜂种的初探．养蜂科技，1995，(6)：19
[78] 王加聪．卡尼鄂拉蜂高产及抗白垩病蜂种的选育．蜜蜂杂志，2005，5：3～5
[79] Keryn Wilkes & Dr. Ben Oldroyd. Breeding hygienic disease resistant bees. A report for the Rural Industries Research and Development Corporation, 2002
[80] Flores JM, Gutierrez I, Puerta F. Oxytetracycline as a predisposing condition for chalkbrood in honeybee. Vet Microbiol, 2004,103 (3-4):195～204
[81] 冯峰等．多功能生物制剂综合防治蜜蜂疾病中间试验．蜜蜂杂志，2002，6：4～5
[82] Martha A. Gilliam. Microbes Help Bees Battle Chalkbrood. Agricultural Research, 1998,8:16～17
[83] Dr Craig Davis and Wendy Ward. Control of chalkbrood disease with natural products. A report for the Rural Industries Research and Development Corporation, 2003,12:3～4
[84] F. J. Reynald et al. Inhibition of the growth of Ascosphaera apis by Bacillus and Paenibacillus strains isolated from honey. Revista Argentina de Microbiologia, 2004,36:52～55
[85] Mourad AK, Zaghloul OA, et al. A novel approach for the management of the chalkbrood disease infesting honeybee Apis mellifera L. (Hymenoptera: Apidae) colonies in Egypt. Commun Agric Appl Biol Sci, 2005,70(4):601～612
[86] 李旭涛．中草药防治白垩病初报．养蜂科技，1993，6：11～12
[87] 高先沛．中草药防治蜜蜂病虫害药方举例．蜜蜂杂志，2005，10：28～29
[88] 赵磊，杨永红等．白垩病研究进展．中兽医医药杂志，2006，2：62～64
[89] 胡能书，万贤国．同工酶技术及其应用．湖南科学技术出版社，1985
[90] 白海艳，史丽，铁军．同工酶电泳技术在昆虫分类学研究中的应用，晋东南师范专科学校学报，2004，21(5)：36～39
[91] 徐忠东，肖家军．同工酶研究及其应用进展．安徽教育学院学报，2000，18(3)：43～45
[92] Frohlich DR, Brindley WA, Burris TE, et al. Esterase isozymes in a solitary bee, Megachile rotundata (Fab.): characterization, developmental multiplicity, and adult variability. Biochem Genet. 1990,28(7-8):347～404
[93] 童富淡，鲍秀良，陈盛禄．意大利蜜蜂苹果酸脱氢酶Ⅱ同工酶基因型研究．浙江农业学报，1998，10(2)：111～112
[94] 刘艳荷，陈盛禄，童富淡．西方蜜蜂正反交苹果酸脱氢酶Ⅱ基因的遗传差异．农业生物技术学报，2001，9(3)：218～237
[95] 刘艳荷，胥保华．西方蜜蜂6个亚种正反交苹果酸脱氢酶Ⅱ基因的遗传差异．上海交通大学学报(农业科学版)，2003，21(2)：86～90
[96] Spoonamore JE, Frohlich DR, Wells MA. p-Nitrophenylacetate hydrolysis by honey bee esterases: kinetics and inhibition. Xenobiotica, 1993, 23(3):279～362
[97] 郭晓霞，郑哲民，于广志．酯酶同工酶多态性及其在昆虫分类学中的应用价值．昆虫知识，2000，37(6)：371～374
[98] 何士敏，肖静，白士博．蜜蜂酯酶同工酶的比较研究．生物技术，2005，15(1)：22～24
[99] 童富淡，汪俏梅等．西方蜜蜂四个亚种酯酶同工酶型和苹果酸脱氢酶Ⅱ同工酶基因型的遗传差异．动物学报，2002，48(6)：828～832
[100] 刘艳荷，陈盛禄，张传溪．西方蜜蜂苹果酸脱氢酶Ⅱ等位基因频率与经济性状的相关性．上海交通大学学报(农业科学版)，2006，24(1)：79～83
[101] E. P. B. Contel, M. A. Mestriner and E. Martins. Genetic control and developmental expression of malate dehydrogenase in Apis mellifera. Biochemical Genetics, 1977, 15(9): 859～876
[102] M. F. P. S. Machado and E. P. B. Contel. Glycerol-3-phosphate dehydrogenase isozyme variation in adult meliponids (Hymenoptera: Apidae). Biochemical Genetics, 1991, 29 (11): 593～600
[103] 刘艳荷，张传溪等．蜜蜂(Apis)苹果酸脱氢酶同工酶的研究进展．遗传，2002，24(5)：586～59
[104] 刘艳荷，陈盛禄等．西方蜜蜂六个亚种苹果酸脱氢酶Ⅱ基因的遗传差异．昆虫学报，2002，45(2)：188～192
[105] 孙亮先，陈朝阳等．四个意大利蜜蜂品系苹果酸脱氢酶Ⅱ基因的遗传差异分析．漳州师范学院学报(自然科学版)，2004，17(1)：53～59
[106] Mahmoud Rouabhia, 2Martin Schaller, et al. Virulence of the Fungal Pathogen Candida albicans Requires the Five Isoforms of Protein Mannosyl transferases. INFECTION AND IMMUNITY, 2005,73(8):4571～4580
[107] 陈伟群，张天宇．十字花科上的链格孢属真菌同工酶凝胶电泳分析．真菌学报，1994，13(4)：295～302
[108] 刘伟成等．球壳孢目真菌同工酶电泳研究Ⅰ．分类学意义的评价．沈阳农业大学学报，2001，32(2)：102～106
[109] 刘伟成等．球壳孢目真菌同工酶电泳研究Ⅱ Ascochyta等八属代表种的分析．吉林农业大学学报，2002，24(1)：47～52
[110] 朴美兰，仝振祥，王凤成．柞蚕不同品种酯酶同工酶的特异性．北方蚕业，2005，(2)：28～30
[111] 汪家政，范明．蛋白质技术手册．北京：科学出版社，2000
[112] 郭尧君．蛋白质电泳实验技术．北京：科学技术出版社，1999
[113] 陆健．蛋白质纯化技术及应用．北京：化学工业出版社，2005
[114] 余来宁，方耀林等．乳酸脱氢酶与酯酶同工酶同板染色法．生物化学与生物物理进展，1998，25(5)：462～464
[115] 马雅军，王菊生．赫坎按蚊种团中5种4株的数值分类．贵阳医学院学报，1990．15(1)：45～48
[116] Hyeon-Su Ro, Hyung Pyo Hong, et al. Genome-wide cloning and characterization of microbial esterases. FEMS Microbiology Letters, 2004,233:97～105
[117] Li Shao-wen, Meng Yu-ping, et al. A comparative study of esterase isozymes in 6 species of Apis and a genera of Apoidea. Journal of Apicultural Research, 1986,25(3):129～136
[118] 尹俊梅，欧文军，张欣．三角酶品种间亲源关系的蛋白质分析．热带作物学报，2001，22(4)：61～66
[119] Baldw in JE and H Krebs. The evolution of metabolic cycles. Nature, 1981,291:381～382
[2] 徐伟松，李建丰，胡美英．昆虫病原真菌在害虫防治上的应用．广西农业科学，2003，6：51～52
[3] 胡奇，刘戬，闫妍．昆虫病原真菌研究进展．天津农学院学报，2004，11(4)：46～51
[4] Iskandarov US, Guzalova AG, Davranov KD. Effects of nutrient medium composition and temperature on the germination of conidia and the entomopathogenic activity of the fungi. Prikl Biokhim Mikrobiol, 2006, 42(1): 81～86
[5] Xu CP, Sinha J, et al. Optimization of physical parameters for exo-biopolymer production in submerged mycelial cultures of two entomopathogenic fungi Paecilomyces japonica and Paecilomyces tenuipes. Lett Appl Microbiol, 2006, 42(5): 501～507
[6] 张素芬．国外昆虫病原真菌研究动态．天津农林科技，1997，140(2)：40～42
[7] Cho EM, Liu L, Farmerie W, Keyhani NO. EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. Ⅰ. Evidence for stage-specific gene expression in aerial conidia, in vitro blastospores and submerged conidia. Microbiology, 2006, 152(9): 2843～2897
[8] Cho EM, Boucias D, Keyhani NO. EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. Ⅱ. Fungal cells sporulating on chitin and producing oosporein. Microbiology, 2006, 152(9): 2855～2919
[9] 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．中国生物防治，1998，14(3)：123～129
[10] Crespo R, Pedrini N, et al. Volatile organic compounds released by the entomopathogenic fungus Beauveria bassiana. Microbiol Res, 2006, 27
[11] 王成树，李增智．虫生真菌分子生物学研究进展．微生物学通报，2001，28(3)：88～92
[12] No authors. The effect of entomopathogenic fungi on the activity of detoxicating enzymes of larvae of bee pyralid Galleria mellonella L. (Lepidoptera, Pyralidee) and the role of detoxicating enzymes in the formation of the insect' resistance to entomopathogenic fungi. Izv Akad Nauk Ser Biol, 2006, (6): 712～720
[13] Pedrini N, Crespo R, Juarez MP. Biochemistry of insect epicuticle degradation by entomopathogenic fungi. Comp Biochem Physiol C Toxicol Pharmacol, 2006, 30
[14] 李建庆，张永安等．昆虫病原真菌毒素的研究进展．林业科学研究，2003，16(2)：233～239
[15] ParkNS, Lee KS, etal. Molecular cloning, expression, and characterition of the Cu, Zn superoxide dismutase (SOD1) gene from the entomopathogenic fungus Cordyceps militaris. Mycologia, 2005, 97(1): 130～138
[16] 赵继红，李建中．农用微生物杀菌剂研究进展．农药，2003，42(5)：6～8
[17] 张丽萍，张贵云．微生物农药研究进展．北京农业科学，2000，18(4)：22～24
[18] 裴炎，方卫国，张永军．昆虫病原真菌致病寄主的机制和基因工程改良．农业生物技术学报，2003，11(3)：221～226
[19] 李勇，杨慧敏．微生物农药的研究和应用进展．贵州农业科学，2003，31(2)：62～63
[20] 周传恩．我国杀虫微生物的应用研究进展及发展前景．农药，2001，40(7)：8～10
[21] Hermann Strasser, Alain Vey, Tariq M. Butt. Are There any Risks in Using Entomopathogenic Fungi for Pest Control, with Particular Reference to the Bioactive Metabolites of Metarhizlum, Tolypocladium and Beauverla species?. Biocontrol Science and Technology, 2000, 10(6):717～735
[22] Cuthbertson AG, North JP, Walters KF. Effect of temperature and host plant leaf morphology on the efficacy of two entomopathogenic biocontrol agents of Thrips palmi (Thysanoptera: Thripidae).Bull Entomol Res, 2005,95(4):321～327
[23] Lee SY, Nakajima I, et al. Cultivation of entomopathogenic fungi for the search of antibacterial compounds. Mycopathologia, 2005, 160(4):321～326
[24] 林超，孙辉等．菌寄生真菌的研究进展．菌物研究，2005，3(1)：46～52
[25] Marannino P, Santiago-Alvarez C, et al. A new bioassay method reveals pathogenicity of Metarhizium anisopliae and Beauveria bassiana against early stages of Capnodis tenebrionis (Coleoptera; Buprestidae). J Invertebr Pathol, 2006,93(3):210～213
[26] Majchrowicz, I; Poprawski, TJ. Effects in vitro of nine fungicides on growth of entomopathogenic fungi. Biocontrol Science and Technology, 1993,3(3):321～336
[27] 李多川．菌寄生真菌分子生物学研究进展．吉林农业大学学报，1998，20：37～39
[28] 邢晓科，郭顺星．菌寄生真菌与寄主相互作用的生化研究进展．菌物系统，2002，21(1)：141～146
[29] 陈大福，梁勤．蜜蜂白垩病的研究进展(连载一)．蜜蜂杂志，2001，1：3～4
[30] 李真银．中西药结合防治蜜蜂白垩病．中国兽医杂志，1994，10：37
[31] 朱亮光，刘嵋恩，何铠光．台湾蜜蜂白垩病发生之初报[J]．台湾大学植物病虫害，1984，(11)：5～7
[32] 董秉义．白垩病的诊断．中国养蜂，1992，(2)：39—40
[33] 陈淑静，康雪冬，冯峰．蜜蜂白垩病研究初报[J]．中国养蜂，1994，(6)：4～7
[34] Zaghloul OA, Mourad AK, El Kady MB et al. Assessment of losses in honey yield due to the chalkbrood disease, with reference to the determination of its economic injury levels in Egypt. Commun Agric Appl Biol Sci, 2005,70(4):703～717
[35] 郭志儒．中华人民共和国农业部第53号令公布动物病原微生物分类名录．中国兽医学报，2005，25(5)：473
[36] X. Qin, J. D. Evans, et al. Genome sequences of the honey bee pathogens Paenibacillus larvae and Ascosphaera apis Insect. Molecular Biology, 2006, 15(5):715～718
[37] 王建鼎，梁勤，苏荣．蜜蜂保护学．中国农业出版社，1997
[38] 邵瑞宜．蜜蜂白垩病病原观察及其综合防治措施．福建农林大学学报，1997，26(3)：345～348
[39] 余林生，孟祥金．蜜蜂白垩病的病原和防治技术研究．中国兽医科技，1999，29(12)：6～8
[40] Heath L A F. Chalkbrood pathogen, A review. Bee World, 1982,63(4): 130～135
[41] Gilliam M. Infectivity and survival of the chalkbrood pathogen, Ascosphaera apis in colonies of honeybee Apis mellifera. Apidologie, 1986,17:93～100
[42] Liang kuang Chu, Thomas Meien Liu, Kai kuang Ho. Growth inhibition of Ascosphaera apis by royal jelly and hydroxydecenoic acid, Reprinted from Bulletin of the Institute of Zoology. Academia sinica, 1992,31(2):73～79
[43] Lu R, Goerzen DW et al. Use of RhPD analysis for in situ identification of Ascosphaera aggregata and Ascosphaera larvis in larval cadavers of the alfalfa leafcutting bee, Megachile totundata. J Invertebr Pathol, 1996,68(1):78～83
[44] R. R. James and J.S. Skinner. PCR diagnostic methods for Ascosphaera infections in bees. Journal of Invertebrate Pathology, 2005,90:98～103
[45] 梁勤，陈大福，王建鼎．温度、相对湿度和pH对蜜蜂球囊菌孢子萌发的影响．应用生态学报，2000，11(6)：869～872
[46] James RR, Buckner JS. Lipids stimulate spore germination in the entomopathogenic ascomycete Ascosphaera aggregata. Mycopathologia, 2004, 158(3):293～302
[47] 牟方喆．蜂蜜中蜜蜂子囊球菌检测规范．吉林粮食高等专科学校学报，1999，14(4)：7～12
[48] 余林生，孟祥金等．蜜蜂白垩病发病规律与防治措施的研究．养蜂科技，1999，3：4～7
[49] 黄通明．蜜蜂白垩病的治疗．养殖天地，2002，148(6)：26
[50] Pawet Chorbinski. The development of the infection of Apismellifera larvae by Ascosphaora apis. Electronic Journal of Polish Agricultural Universities, Veterinary Medicine, 2004, 7(2)
[51] 王志，薛运波．试论蜂场生态与蜜蜂病敌害发生的关系．中国养蜂，2005，56(3)：23
[52] 时丽君．蜜蜂白垩病发病规律及防治措施．吉林畜牧兽医，2003，12：45～46
[53] 张华瑞．试述蜜蜂白垩病的流行特点与致病因素．养蜂科技，1994，4：16～17
[54] R. R. James and T. L. Pitts - Singer. Ascosphaera aggregata contamination on alfalfa leafcutting bees in a loose cell incubation system [J]. Journal of Invertebrate Pathology, 2005,89(2):176～178
[55] John. D. Vandenberg. Bioassay of the chalk brood fungus Ascosphaera aggregata on larvae of the alfalfa leafcutting bee, Megachile rotundata[J]. Journal of Invertebrate Pathology, 1992,60(2):159～163
[56] John D. Vandenberg, Becy L. Fichter, and W. P. Stephen. Spore Load of Ascosphaera Species on Emerging Adults of the Alfalfa Leafcutting Bee, Megachile rotundatat. Applied and Environmental Microbiology, 1980,39(3): 650～655
[57] 范正友．蜜蜂白垩病流行病学研究进展．中国养蜂，1996，132(1)：15～16
[58] Flores JM, Gutierrez I, Espejo R. The role of pollen in chalkbrood disease in Apis mellifera: transmission and predisposing conditions. Mycologia, 2005,97(6):1171～1176
[59] Flores JM, Spivak M, Gutierrez I. Spores of Ascosphaera apis contained in wax foundation can infect honeybee brood. Vet Microbiol, 2005, 108(1-2):141～145
[60] 牛庆生．长白山区蜜蜂白垩病及其防治[J]．吉林畜牧兽医，1997，(7)：36
[61] 李怀军．浅析呼盟地区白垩病复发的因素．蜜蜂杂志，2005，6：23～24
[62] 李真银．中西药结合防治蜜蜂白垩病．中国兽医杂志，1994，10：37
[63] J. M. Flores, I. Gutierrez and F. Puerta. Oxytetracycline as a predisposing condition for chalk brood in honey bee [J]. Veterinary Microbiology, 2004, 103(3):195～199
[64] Martha Gilliam. Identification and roles of non-pathogenic microflora associated with honey bees[J]. FEMSMicrobiology Letters, 1997,155(1):1～10
[65] 吴殿军，吴永茂等．蜜蜂检疫和二类传染病的检疫处理．广西畜牧兽医，2005，21(5)：234～235
[66] 王桂忠．蜜蜂白垩病的检疫报告．宁夏农学院学报，1997，18(2)：88～89
[67] 郑祥义，李真银．蜜蜂白垩病综合防治初探．养蜂科技，2002，4：14～15
[68] Anderson DL; Giacon H; Gibson NL. Culture, detection and thermal destruction of the chalkbrood fungus, Ascosphaera apis. Journal of Apicultural Research, 1997,36:163～168.
[69] James RR. Impact of disinfecting nesting boards on chalkbrood control in the alfalfa leafcutting bee. J Econ Entomol, 2005,98(4):1094～1100
[70] 李怀军．防治白垩病关键在消毒．蜜蜂杂志(月刊)，2005，12：28
[71] 陈大福，梁勤．蜜蜂白垩病的研究进展(连载二)．蜜蜂杂志，2001，2：10～13
[72] 王福仁．白垩病的综合防治方法．中国养蜂，1995，2：11
[73] 陈东海，历延芳等．谈长白山区蜜蜂白垩病发病率低的因素．中国养蜂，1996，5：17
[74] Tarpy DR, Seeley TD. Lower disease infections in honeybee (Apis mellifera) colonies headed by polyandrous vs monandrous queen. Naturwissenschaften, 2006, 93(4): 195～204
[75] 王志．蜜蜂种群习性与蜂病流行的关系．养蜂科技，20047，3：22～23
[76] 罗勤．抗白垩病蜂群的选育实践．蜜蜂杂志，2001，11：22～23
[77] 徐家胃．培育抗白垩病蜂种的初探．养蜂科技，1995，(6)：19
[78] 王加聪．卡尼鄂拉蜂高产及抗白垩病蜂种的选育．蜜蜂杂志，2005，5：3～5
[79] Keryn Wilkes & Dr. Ben Oldroyd. Breeding hygienic disease resistant bees. A report for the Rural Industries Research and Development Corporation, 2002
[80] Flores JM, Gutierrez I, Puerta F. Oxytetracycline as a predisposing condition for chalkbrood in honeybee. Vet Microbiol, 2004,103 (3-4):195～204
[81] 冯峰等．多功能生物制剂综合防治蜜蜂疾病中间试验．蜜蜂杂志，2002，6：4～5
[82] Martha A. Gilliam. Microbes Help Bees Battle Chalkbrood. Agricultural Research, 1998,8:16～17
[83] Dr Craig Davis and Wendy Ward. Control of chalkbrood disease with natural products. A report for the Rural Industries Research and Development Corporation, 2003,12:3～4
[84] F. J. Reynald et al. Inhibition of the growth of Ascosphaera apis by Bacillus and Paenibacillus strains isolated from honey. Revista Argentina de Microbiologia, 2004,36:52～55
[85] Mourad AK, Zaghloul OA, et al. A novel approach for the management of the chalkbrood disease infesting honeybee Apis mellifera L. (Hymenoptera: Apidae) colonies in Egypt. Commun Agric Appl Biol Sci, 2005,70(4):601～612
[86] 李旭涛．中草药防治白垩病初报．养蜂科技，1993，6：11～12
[87] 高先沛．中草药防治蜜蜂病虫害药方举例．蜜蜂杂志，2005，10：28～29
[88] 赵磊，杨永红等．白垩病研究进展．中兽医医药杂志，2006，2：62～64
[89] 胡能书，万贤国．同工酶技术及其应用．湖南科学技术出版社，1985
[90] 白海艳，史丽，铁军．同工酶电泳技术在昆虫分类学研究中的应用，晋东南师范专科学校学报，2004，21(5)：36～39
[91] 徐忠东，肖家军．同工酶研究及其应用进展．安徽教育学院学报，2000，18(3)：43～45
[92] Frohlich DR, Brindley WA, Burris TE, et al. Esterase isozymes in a solitary bee, Megachile rotundata (Fab.): characterization, developmental multiplicity, and adult variability. Biochem Genet. 1990,28(7-8):347～404
[93] 童富淡，鲍秀良，陈盛禄．意大利蜜蜂苹果酸脱氢酶Ⅱ同工酶基因型研究．浙江农业学报，1998，10(2)：111～112
[94] 刘艳荷，陈盛禄，童富淡．西方蜜蜂正反交苹果酸脱氢酶Ⅱ基因的遗传差异．农业生物技术学报，2001，9(3)：218～237
[95] 刘艳荷，胥保华．西方蜜蜂6个亚种正反交苹果酸脱氢酶Ⅱ基因的遗传差异．上海交通大学学报(农业科学版)，2003，21(2)：86～90
[96] Spoonamore JE, Frohlich DR, Wells MA. p-Nitrophenylacetate hydrolysis by honey bee esterases: kinetics and inhibition. Xenobiotica, 1993, 23(3):279～362
[97] 郭晓霞，郑哲民，于广志．酯酶同工酶多态性及其在昆虫分类学中的应用价值．昆虫知识，2000，37(6)：371～374
[98] 何士敏，肖静，白士博．蜜蜂酯酶同工酶的比较研究．生物技术，2005，15(1)：22～24
[99] 童富淡，汪俏梅等．西方蜜蜂四个亚种酯酶同工酶型和苹果酸脱氢酶Ⅱ同工酶基因型的遗传差异．动物学报，2002，48(6)：828～832
[100] 刘艳荷，陈盛禄，张传溪．西方蜜蜂苹果酸脱氢酶Ⅱ等位基因频率与经济性状的相关性．上海交通大学学报(农业科学版)，2006，24(1)：79～83
[101] E. P. B. Contel, M. A. Mestriner and E. Martins. Genetic control and developmental expression of malate dehydrogenase in Apis mellifera. Biochemical Genetics, 1977, 15(9): 859～876
[102] M. F. P. S. Machado and E. P. B. Contel. Glycerol-3-phosphate dehydrogenase isozyme variation in adult meliponids (Hymenoptera: Apidae). Biochemical Genetics, 1991, 29 (11): 593～600
[103] 刘艳荷，张传溪等．蜜蜂(Apis)苹果酸脱氢酶同工酶的研究进展．遗传，2002，24(5)：586～59
[104] 刘艳荷，陈盛禄等．西方蜜蜂六个亚种苹果酸脱氢酶Ⅱ基因的遗传差异．昆虫学报，2002，45(2)：188～192
[105] 孙亮先，陈朝阳等．四个意大利蜜蜂品系苹果酸脱氢酶Ⅱ基因的遗传差异分析．漳州师范学院学报(自然科学版)，2004，17(1)：53～59
[106] Mahmoud Rouabhia, 2Martin Schaller, et al. Virulence of the Fungal Pathogen Candida albicans Requires the Five Isoforms of Protein Mannosyl transferases. INFECTION AND IMMUNITY, 2005,73(8):4571～4580
[107] 陈伟群，张天宇．十字花科上的链格孢属真菌同工酶凝胶电泳分析．真菌学报，1994，13(4)：295～302
[108] 刘伟成等．球壳孢目真菌同工酶电泳研究Ⅰ．分类学意义的评价．沈阳农业大学学报，2001，32(2)：102～106
[109] 刘伟成等．球壳孢目真菌同工酶电泳研究Ⅱ Ascochyta等八属代表种的分析．吉林农业大学学报，2002，24(1)：47～52
[110] 朴美兰，仝振祥，王凤成．柞蚕不同品种酯酶同工酶的特异性．北方蚕业，2005，(2)：28～30
[111] 汪家政，范明．蛋白质技术手册．北京：科学出版社，2000
[112] 郭尧君．蛋白质电泳实验技术．北京：科学技术出版社，1999
[113] 陆健．蛋白质纯化技术及应用．北京：化学工业出版社，2005
[114] 余来宁，方耀林等．乳酸脱氢酶与酯酶同工酶同板染色法．生物化学与生物物理进展，1998，25(5)：462～464
[115] 马雅军，王菊生．赫坎按蚊种团中5种4株的数值分类．贵阳医学院学报，1990．15(1)：45～48
[116] Hyeon-Su Ro, Hyung Pyo Hong, et al. Genome-wide cloning and characterization of microbial esterases. FEMS Microbiology Letters, 2004,233:97～105
[117] Li Shao-wen, Meng Yu-ping, et al. A comparative study of esterase isozymes in 6 species of Apis and a genera of Apoidea. Journal of Apicultural Research, 1986,25(3):129～136
[118] 尹俊梅，欧文军，张欣．三角酶品种间亲源关系的蛋白质分析．热带作物学报，2001，22(4)：61～66
[119] Baldw in JE and H Krebs. The evolution of metabolic cycles. Nature, 1981,291:381～382