摘要
玉米鼠耳病(Maize wallaby ear virus MWEV)是我国近年来发生的一种重要玉米病害，具有毁灭性、流行性、爆发性等特点。二点叶蝉Cicadulina bipunctella(Mats)是玉米鼠耳病的主要介体昆虫之一，在该病的发生、扩展蔓延中起着关键作用。因此，我们着眼于西南地区玉米鼠耳病发生状况，在确定玉米鼠耳病优势介体种群的基础上，探讨二点叶蝉与玉米鼠耳病的关系，建立实验种群，研究环境因素尤其是温度对二点叶蝉种群的影响。同时对玉米鼠耳病病原进行鉴定，以期确定玉米鼠耳病病原；用提纯的病原制备抗血清，建立玉米鼠耳病检测系统。研究结果如下：
1．二点叶蝉和玉米鼠耳病关系的确立
在玉米鼠耳病病区采集为害玉米的主要昆虫，对采集的昆虫的进行传毒测定，结果表明仅二点叶蝉能够致使玉米表现出鼠耳病的典型症状，且二点叶蝉是玉米鼠耳病病区为害玉米的优势叶蝉种群(平均密度为0.0629～3.0114头/株)。玉米被二点叶蝉取食后，其发病率高达96％。该病表现为系统性矮化，叶片皱缩粗糙，僵直挺立，症状类似玉米粗缩病。其余的飞虱、叶蝉、蚜虫及蝽类取食玉米后不能导致玉米出现典型的玉米鼠耳病症状。证明二点叶蝉是玉米鼠耳病的介体昆虫之一。
2．玉米鼠耳病传毒方式及毒源的繁殖
玉米鼠耳病不能通过摩擦接种、菟丝子桥接、注射接种的方式传播，玉米鼠耳病毒源的建立与繁殖只能依靠二点叶蝉传毒完成。玉米最适于二点叶蝉种群增长，且玉米植株上症状明显，种植方便，生长迅速，是毒源繁殖的最佳寄主。将带毒的二点叶蝉转到N.tabacum somsun(n/n)，N. samsun(N/N), N. tabacum xamthi(nc)，K326，白肋烟，心叶烟，普通烟，本氏烟，珊西烟，苋色黎，昆黎，灰黎，黄苗榆，矮牵牛，番茄，假酸浆上后，二点叶蝉不能在这些双子叶植物上存活，且上述植物无症状出现，因此不能作为玉米鼠耳病病毒的鉴别寄主。
以玉米为寄主，在实验室成功地建立了二点叶蝉实验种群并繁殖了大量的玉米鼠耳病病株毒源，在25～28℃条件下，二点叶蝉在实验室大约30天一代，己连续饲养了20代。每个养虫箱内二点叶蝉成虫大约在3000头左右，共收集到约5kg的玉米鼠耳病病株。
3．无毒种群的建立
分别采用从田间采集、从卵分离、蜕皮分离、混合种群分离的方法建立二点叶蝉无毒种群。结果表明，二点叶蝉自然混合种群利实验种群中不存在无毒种群。在田间，二点叶蝉是鼠耳病存在、发病和蔓延的前提，玉米鼠耳病为二点叶蝉的存在提供基本信息。二点叶蝉取食24h时，虽部分个体不能致玉米发生鼠耳病。但是对其子代进行致病测试实验表明，该种
西南农业人学硕[学位论文
中文摘要
群仍然可以致玉米表现出鼠耳病病症，且发病率高达94.5%，与原始种群发病率相似。
4.温度对二点叶蝉的发育、存活及繁殖的影响
系统研究了不同温度(巧，20，25，28，犯，35℃)条件下二点叶蝉实验种群在玉米幼苗
上的发育、存活情况，并用生命表资料分析了温度对其种群增长的影响，拟合二点叶蝉发育
速率与温度之间的关系模型，求出各发育阶段的发育起点温度和发育上限、下限温度，组建
不同温度下实验种群特定时间生命表，计算种群增长参数，分析种群增长参数与温度之间的
关系。结果表明，在巧℃下，卵不能完成发育，在35℃条件下三龄若虫不能存活。二点叶蝉种
群增长最适温区为25一32℃。在该温区范围内，内票生殖力为0.0631一0.0463，周限增长率为
】.0561一1 .0497，种群存活曲线属于Deeveyl型，而在15℃不利条件下，种群存活曲线属于Deevey
川型
5.玉米鼠耳病病原鉴定
对二点叶蝉唾液腺，马氏管，肠道以及玉米病叶，病根，耳突组织超薄切片透射电镜观
察，在二点叶蝉的肠道切片上观察到疑是玉米鼠耳病的病毒粒子，玉米病叶、病根、耳突组
织上未能观察到病毒粒子。优化病毒提纯程序，基本提纯了玉米鼠耳病病毒，其分光光度计
检测紫外吸收值A26办280=2 .1。根据提纯的病毒液260nm处的吸光值，计算出提纯病毒的浓
度为1.6m留m!，产量为0.05u留g病株材料。用四环素等抗生素类药剂处理病株，结合玉米病
株、二点叶蝉各组织的超薄切片，电镜观察结果排除了植原体的可能性。根据病害的病症、
传播介体，以及病原的初步鉴定结果把发生在四川南充，重庆壁山的玉米病害诊断为玉米鼠
耳病，玉米鼠耳病病原疑为玉米鼠耳病毒(MWEV)。
6.玉米鼠耳病检测系统的建立
用玉米鼠耳病病株分离提纯了玉米鼠耳病病毒，制备了特异性的抗血清。利用价格低廉
的羊抗兔酶标抗体，建立了快速灵敏的OAS一ELISA检测方法。通过DAS一ELISA方阵实验，
确定抗血清及羊抗兔酶标抗体的稀释度分别是l:1000和1:5000，以此作为DAs一ELISA检
测的最适合条件。DAS一ELISA检测病叶粗提纯液的最大稀释度可以达到1:640。在实际中我
们选用1:20作为病叶粗提纯液的稀释度，此时阳性反应0D值较高，阴阳反应差异较大。将
建立的DAS一ELISA检测方法用来检测带毒的_一点叶蝉时，其检测结果不理想(0D值仅为
0 .574)。
Maize wallay ear virus (MWEV), a kind of important maize disease firstly burst out in Sichuan province at the end of 20th century, was caused by(Cicadulina bipunctella (mats) . It damages maize in field and leads to great economic losses when the population occurs in abundance. The dominance population is ascertained by the field investigations and indoor trials, the relationships between C bipunctella and MWEV, the bio-ecological characteristics of C. bipunctella, were studied systematically. The virus was identified as MWEV, The DAS-ELISA method for MWEV detection was developed using Antiserum prepared by the purified virus and antibody, The research contents and results were listed as follows: l.The relation between MWEV and Cicadula. bipunctella (mats)
The insects that collected from the MWEV disease field were transmitted to the corn. The result shows the C. bipunctella (mats) can induce the maize to the symptom of dwarf, crimpled leaf just like the symptom of MWEV. And the C. bipunctella (mats) population is the dominance leafhopper population in the field (0.0629-3.0114 per corn). And the incidence of disease was 98% by this insect. The other insects can not induce the maize to this symptom. All of this result shows that the C. bipunctella (mats) was one of the vector of MWEV.
2.The propagate of MWEV
The MWEV can't be transmitted by graft, cuscuta australis R.Br, injecting while can transmitted by C. bipunctella (mats). The construction and propagation of MWEV depend on the C. bipunctella (mats). Zea mays L was conformed to the adaptive host for the leafhopper for the development, survivorship, longevity, propagation of C. bipunctella (mats) on the six host: Zea mays L. Triticum aestivum L. Sorghum vulgare Pers. Oryza saliva L. Setaria italica (L.) Beauv. Hordeum vulgare L and the maize were easy to plant, growth rapidly, the symptom obvious. The results shows the C. bipunctella dead in the 3-5 days After C. bipunctella with MWEV is transferred to N.Tabacum samsun (n/n), N.samsun (N/N), N. tabacum xamthi (nc), K326, N. tobacum White Burly, N. glutinosa, Nicotiano tabacum L. Nicotiana tabacum cv.Xanthi-nc, chenopodium amaranticolor, C. quinoa, .album, N.rustica, Peturnia hybrida, Lycopersicon escultentum., Nicandra physaioides as respectively. And all of the plant haven't any different with the blank. So
all these dicotyledonous can't differentiate for MWEV and the host for MWEV.
The leafhopper population were successfully constructed in the lab by maize and supplied by
the natural population and disease plant timing, under 25-28*C.the . bipunctella (mats) population were reared to 20 generation, 3000 per cultivated box. 5kg material were Gathered.The propagation of MEEV, transmit, entomological biology were guaranteed.
3.The construction of C.bipunctella.(mats) of free virus
Collecting from the field, separate from eggs, separate from ecdysis, separate through mixed leafhopper population were used to constructed the population of C.bipuncte!la.(mats) free from MWEV. In the field the C.bipunctella.(mats) was the precondition of the existing, occurring and spread of MWEV .and the MWEV indicating the existing of C.bipunctella.(mats). 24h after the C.bipunctella.(mats) was transfer to the maize, some of leafhopper can not transfer the virus.but it's offspring still have the ability to transmit MWEV, and almost same with the original population , All of the methods are unsuccessful. The population of C.bipunctella.(mats) collecting from the disease field were feed by maize, supply with the nature population or maize with MWEV timing. The experiment population that constructed by this method has similar behavior and transmit virus characteristic with the nature population.
4.Effect of Temperature on of C bipunctella (mats)
Development survivorship, longevity, and reproduction of Cicadulina bipunctella (mats) was measured at 15,20,25,28,32,35℃,.the population reared at 35℃ failed to develop. The population rear at 32℃,had the highest intrinsic rate of increase (0.0542),higher net reproduc
引文
1 .Tryon, H. "Report of the Department of Agriculture and Stock, Queesland for 1909-1910". 1910. 81-82.
2. Simmonds. J.H. "Report of the Department of Agriculture and Stock, Queesland for 1937-1938". 1938.36.
3. Schindler, A, J. Insect transmission of"wallaby ear" disease of maize. Journal of the Australian Institute of Agricultural Science. 1942.(8):35-37.
4. Grylls-N.E. Leafhopper transmission of a virus causing maize wallaby ear disease. Annals-of-Applied-Biology. 1975.79: 3,283-296; 2 pl.; 36 ref.
5. Boccardo-G; Hatta-T; Francki-RIB; GrivelI-CJ. Purification and some properties of reovirus like particles from leafhoppers and their possible involvement in wallaby ear disease of maize. Virology. 1980.100: 2, 300-313; 6 fig.; 27 ref.
6. Hatta, T., Boccardo, G., Francki, R.I.B. Anatomy of leaf galls induced by Reoviridae and by wallaby ear disease Fijivirus, maize, Laodelphax striatellus. Physiol-Plant-Pathol. London, Academic Press, 1982.20 (1): 43-46.
7. Ofori, F.A., Francki, R.I.B. 1983. Evidence that maize wallaby ear disease is caused by an insect toxin Leafhopper Cicadulina bimaculata Evans. Ann-Appl-Biol. London: Association of Applied Biologists. 103 (2): 185-189.
8．吕宏声．昆虫病毒分子生物学．中国农业科学出版社．北京．1998．p415．
9．邓先明．玉米粗缩病(MRDV)和鼠耳病(MWEV)的发生和研究简况．国外农学—植物保护，1994，7(3-4)：15一18．
10．李祥坤．邓先明，梁映鸿，朱文柄，刘光珍．玉米鼠耳病传毒叶蝉研究。西南农业大学1996．vol(18)6-8．
11．林代福，彭丽娟等．玉米鼠耳病病状识别与发病因素调查．山地农业生物学报，2000，19(4)：262-265．
12. Reddy-DVR; Grylls-NE; Black-LM. Electrophoretic separation of dsRNA genome segments from maize wallaby ear virus and its relationship to other phytoreoviruses. Virology. 1976.73: 1, 36-42; 3 fig., 2 graphs, 1 tab.; 26 ref.
13．南充市玉米病毒病防治研究协作组等．1998．南充市玉米鼠耳病发生规律和防治研究．
14. Hatta-T; Francki-RIB. Virology. Enzyme cytochemical identification of single-stranded and double-stranded RNAs in virus-infected plant and insect cells. 1978.88: 1, 105-117; 9 fig., 1 tab.; 30 ref.
15．全国水稻病毒病研究协作组．1984．我国水稻黄矮病(RYSN)和矮缩病(RDV)的综合防治．中国农业科学3：75——78．
16．广东农林学院植病教研组．水稻黄矮病在广东的发生流行规律及防治经验．16—23页．《水稻矮缩病防治研究资料选编》(浙江农业科学院情报资料室)．1973．148．
17 A．J吉布斯，B．D．哈里森著．1980．植物病毒学概要．上海科学技术出版社．
18．裘维蕃主编．1984．植物病毒学．科学出版社．
19．蔡平，何俊华．2000．植物病原介体叶蝉种类及其传毒特点与传毒方法．华东昆虫学报，9(1)：96-10
20. Chiykowski, L.N.. Leafhopper transmitted pathogens and their vectors. In Plant Disease and Vectors: Ecology and Epidemiology. Ed. K. Maramorsch and K.F. Harrris,. 1981. pp: 131-142.
21．田波，裴美云编．植物病毒研究方法(上册)．科学出版社．1987．
22. Conti. Ed. 1985. Transmission of plant virus by leafhopper and planthopper In: The Leafhopper and Planthoppers. Ed.L.R. Nault and J.G.Rodriguez. pp: 289-307.
23．梁小波，鲁瑞芳，吴云锋，彭学贤．植物病毒昆虫介体传播的研究进展．生物工程进展．2001．(21)：4．
24. Nault, L.R.1989. Leafhopper and planthopper transmission of plant viruses. Ann. Rev. Entomology. 34: 503-529.
25．Takah．，Y，秦文胜．聚合酶链反应检测轻微感染病毒的水稻植株和带毒个体叶蝉内的水稻东格鲁杆状病毒．农业科技译丛(杭州)．1995．(2)．：28-32．
26. Nielson, M.W. 1979. Taxonomec relationships of leafhopper vectors of plant pathogens. In:Leafhopper Vectors and plant disease agents. Ed.K.Maramorsch and K.F.Hariis: pp: 3-27.
27. Goodman.R.M. Geminivirues. In. Handbook of Plant Virus Infection and Comparative Diagnosis Ed. E.Kurstak. New York Elsevier. 1981. 883-910.
28．梁训生，谢联辉．植物病毒学．农业出版社．1994．
29. Diversification at the Insect-plant interface insights from phylogenetics. Brian D.Farrell, Charles Miller, and Douglas J. Futuyma. Bioscience; Jan 1992;42,1; Agricola pg 34.
30 蔡平，何俊华．植物病原介体叶蝉种类及其传毒特点与传毒方法．华东昆虫学报，2000．9(1)：96-102．
31．吴云峰 周广和．非持久性病毒传播机制的研究进展．中国病毒学 1996，11(4)：296-299．
32．吴云峰，魏宁生．非持久性病毒传播专化性研究．西北农业大学学报．1996，5(1)：39-42．
33．于善谦．植物病毒介体传播的分子机制．中国病毒学．1999，14(4)：285-294．
34 蒯元璋，张仲凯，陈海如．我国植物支原体类病害的种类．云南农业大学学报．2000．15(2)：153-160．
35．黄文晋，崔晓江，林木兰，彭学贤．类菌原体研究现状与发展趋势．微生物学报．1994．21(2)：37-40．
36. Davis R.E., E.L.Dally, A.Bertaccini. Cloned DNA probe for specific detection of Italian
periwinkle virescence mycopalsmalike organism (MLOs) and investigation of genetic relatedness with other MLOs. Phytopathol. Mediterr., 1992.31:5-12.
37 Lin.C.P., T.A.Chen. 1985. Monoclonal antibodies against the aster yellows agent. Science, 227:1233-1235.
38 Schigetion N., hiroshi O., Shosuke K., et al. 1993. Phylogenetic diversity of phytopathogenic mycoplasmalike organisms, Int.J.Syst. Bacteriol, 43: 461-467.
39 Clark M.F., A.Morton, S.L.Buss. 1989. Preparation of mycoplasma immunogens from plant and a comparison of polynal association antigen, Ann.Appl.Biol., 114:111-124.
40．戴群，何放亭．桑树萎缩类菌原体的PCR检测．山东大学学报：自然科学版．1997．32(3)：337-341．
41 张锡津，田国忠．温度处理和茎尖培养结合脱除泡桐丛枝病类菌原体(MLO)．林业科学．1994．30(1)：34-38．
42．王焯，陈作义．四环素消毒枣疯病接穗研究．植物保护学报．1992．19(1)：62-62．
43．蔡平，何俊华．中国叶蝉天敌种类及其应用概况(综述)．安徽农业大学．1999．26(1)：74-79．
44．陈常铭．黑肩绿盲蝽的初步研究．植物保护学报．1985．12(1)：69-72．
45 王洪全．我国农田蜘蛛利用研究进展．动物学杂志．1989．24(5)：50-52．
46．彭宇，赵敬钊等．我国关于蜘蛛的研究和利用进展．蛛形学报．1997．6(1)：69-73．
47．郭振中等．贵州农林昆虫志(卷1)．贵州人民出版社．贵阳．1987．
48．安德荣，吴际云，郑文华编著．植物病毒分类和鉴定的原理及方法．陕西科学技术出版社．1995．
49．施曼玲，周雪平．植物病毒病的诊断技术．微生物学通报，2000，27(2)：149-151．
50．马筠．植物病毒鉴定检测方法的研究进展．世界农业．2003．8：50-51．
51．李一农，李芳荣等．建议将拟毛刺线虫属定为潜在危险性线虫．中国检验检疫．2000．(9)：7-7．
52．国际翔，李文清，王丽霞，刘丹，毕庶春．应用免疫电镜技术检测植物病毒的研究．辽宁农业科学．1989．(4)：35-41．
53．林均安，高锦梁，洪建．实用生物电子显微术．沈阳：辽宁科技出版社．1989．
54. Milton Zaithlin, Peter Palukaitis. 2000. Advances in understanding plant viruses and virus diseases. Annual Reviews, 38: 113-127.
55．吴秋豫，陈新华，丁铭等．用二种ELISA法进行的烟草花叶病速诊断试剂盒的研究．华中师范大学学报：自然科学版．2000，34(4)．468-470．
56．薛朝阳，周雪平，青玲等．利用DAS—ELISA进行番茄花叶病毒的田间检测．植物病理学报．1999(29)：157-162．
57．孟清，宋伯符．应用Dot-ELISA检测马铃薯卷叶病毒．植物病理学报．1995．25(1)：61-64．
58. Valkirs. G. E; Barton.R. Immuno concentration a new format for solid phace immunoassays. Clin
Chem,1985.31:1427-1421.
59．徐明全，郑平，刘荣维，刘擎，王韬．植物病毒检测技术-组织印迹法．微生物学通报．2000．27(5)：360-363．
60. Pasquini et al. Detection of plum pox virus in apricot seeds. Acta virologica 1998(42): 260-263.
61．陈炯，程晔．小麦黄花叶病毒和小麦梭条斑花叶病毒的生物学和分子生物学研究．中国病毒学．2000，15(2)：97-105．
62. Lin,N.-S.&Langernberg, W.G., J.Ultrastr.Res. 1984..C9:309-323.
63．胡稳奇．分子生物学技术在植物病霉鉴定与分类中的应用．植物保护．1994，(2)：31-33．
64. M.Belen Suarez, Isabel Grondona et al. Characterization of beet cecrotic yellowvein furorirus from Spanish sugar beets. Internatl microbial. 1999(2):87-92.
65．宁红，秦蓁．分子生物学技术在检疫性有害生物诊断中的应用．植物检疫．2002，16(2)：98-100．
66．孔宝华，陈海如，常胜军，朱水芳，黄文胜，刘进元．RT-PCR检测李坏死环斑病毒的研究．植物检疫．2000 14(5)：257-260．
67．王明霞．应用PCR微量板杂交法检测植物病毒和类病毒．植物检疫．
68. Thornbarry D W., Hellmann G M., Rhoads R E., and Pirone T P., Purification and characterization of potyvirus helper component Virology 1985 144:260-267.
69. M.Hema, H.S.Savithri, P.Screenivasulu. Comparison of direct binding polymerase chain reaction with recombinant coat protein antibody based dot-blot immunobinding assay and immunocapture-reverse transcription-polymerase chain reaction for the detection of sugarcane streak mosaic virus causing mosaic disease of sugarcane in India. Current Science. 2003,85(12):1774-1778.
70. http:www.cau.edu.cn/agrocbi/periodical/nyswjsxb/nysw2000/000414.htm.
71 黄标．玉米铁甲虫的发生与防治．昆虫知识．1997，34(6)．-331-333
72．裴海英，赵奎军．防治玉米根部害虫缓释复合颗粒剂的研究初报．沈阳农业大学学报．1999，30(3)．-381-382．
73．谢联辉，林奇英，吴组建．植物病毒名称及归属．北京．中国农业出版社．1999．
74．蒋军喜．玉米矮花叶病病原鉴定、检测及外壳蛋白基因研究．浙江大学博士论文．2002．5
75．吴云峰．植物病毒学原理与方法．西安地图出版社．西安．1999．
76．张宏瑞，李正跃．2001．媒介昆虫与烟草病毒病关系的研究．云南农业大学学报，16(3)：231-235．
77．李小珍．刘映红，田艳．六种寄主植物对二点叶蝉生长发育和繁殖的影响．待发表．
78．王洪章．菟丝子的鉴别．天津中医学院学报．2001(20)：48．
79．刘丽莎，张西玲，李萍，王琦．菟丝子发芽特性的研究．《中草药》1994．25(12)643-646．
80．侯明生 覃瑞 湖北省白肋烟病毒病毒源种类鉴定．华中农业大学学报．1998．17(6)：
527-530．
81．李雪燕，刘映红，魏劲，青玲．温度对二点叶蝉的发育存活及繁殖的影响．西南农业大学学报(自然科学版)．2004．，26(1)：35-39．
82．张海林，米竹青，张云智．白纹伊蚊和动脉伊蚊经卵传递登革病毒的研究．中国病毒学．1996．11(3)：230-236．
83．苗琛 兰利琼 张小林等．RSSG58基因在水稻精细胞中的表达．植物生理与分子生物学学报．2003，29(3)．-251-256．
84．卢文岱．2000．SPSS for windows．电子工业出版社．
85. Briere, J. F., P. Pracros, A. Y. Roux, and J. S. Pierre. 1999. A novel model of temperature-dependent development for arthropods. Environ. Entomol. 28: 22-29.
86. Pinder JE. 1978. The Weibull distribution: A new method of summarizing survivorship data. Ecology, 59 (5): 175～179.
87. Deevey, E. S. 1947. Life tables for natural populations. Q.rev. Biol. 22:283-314.
88．吴坤君，陈玉平，李明辉．温度对棉铃虫实验种群生长的影响．生态学报，1980．23(4)：358-367．
89 Birch IC. 1948. The intrinsic rate of natural increase in an insect population. J Anim Ecol, 17: 15-26.
90．丁岩钦．1980．昆虫种群数学生态学原理与应用．科学出版社．
91．陈建明，何家安，何俊华．温度和食物对黑肩绿盲蝽发育、存活和繁殖的影响．昆虫学报，1994．37(1)：63-70．
92. Kaihong Wang and James H. Tsai. Temperature Effect on Development and Reproduction of Silverleaf whitefly (Homoptera:Aleyrodidae). 1996.Ann.Entomol .Soc .Am 89(3):373-384.
93. Schoolfield, R. M., P.J.H. Sharp, and C. E. Magnuson. 1981. Nonlinear regression for biological temperature-dependent rate models based on absolute reaction-rate theory. J.Theor. Biol. 88:719-731.
94. Summers. C. G., Coviello, and A. P. Gutierrez. 1984. Influence of constant temperatures on the development and reproduction of Acyrthosiphon kondoi (Homoptera: Aphididae). Environ. Entomol. 13: 236-242.
95. Lactin, D. J., N. J. Holliday, D. L. Johnson, and R. Craigen. 1995. Improved rate model of temperature-dependent development by arthropods. Environ. Entomol. 24: 68-75.
96. Logan, J. A., D. J. Wallkind, S. C. Hoyt, and L. K. Tanigoshi. 1976. An analytic model for description of temperature dependent rate phenomena in arthropods. Environ. Entomol. 5: 1133-1140.
97．吴建宇，秦西云，王学辉．云南烟草从枝症病害病原的研究．云南省植物病理重点实验室论文集．云南科技出版社．昆明．1998．
98．缪晓辉，马亚利，陈士葆等．ELISA技术的某些改进．上海免疫学杂志．1994．4：229-231