金瓜和白瓜花叶病毒病病原的初步鉴定

崇明是上海出口蔬菜生产基地之一,也是国家级的绿色食品园区之一,具有生产绿色蔬菜商品的生态环境和资源优势.     

豇豆病毒病病原的分子鉴定

为澄清浙江省豇豆病毒病病原及其分类,采用马铃薯Y病毒属通用引物Sprimer扩增了浙江豇豆线状病毒基因组3′－末端序列。同时又根据已知CMV RNA3序列设计引物pCMV-44-63/1200-1181,扩增了混合的CMV－ZJ运动蛋白基因全序列。外壳蛋白氨基酸序列分析表明,产中仅存在一种线状病毒（CV－ZJ）,该病毒与一泰国豇豆蚜传花叶病毒（CABMV）具有最高的同源性（98.6%）,与花生条纹病毒（PStV）、石斛花叶病毒（DeMV0．）、赤豆花叶病毒（AZMV）、黑眼豆豆花叶病毒（BICMV）和菜豆普通花叶病毒（BCMV）分离物间的同源性为88.5%－94.4%,而与其它CABMV分离物的同源性均低于70％。进化树分析表明,PStV、AZMV、DeMV、BlCMV和BCMV为同种异名,应统称为BCMV,而CABMV为另一种病毒;CV－ZJ和泰国CABMV分离物应分类为BCMV豇豆株系。CMV－ZJ的运动蛋白序列分析表明,该分离物属于CMV亚组Ⅰ,与其它分离物氨基酸序列同源性为93.1%－97.8%。     

矮牵牛病毒病的初步研究

矮牵牛是一种草本花卉,主要用来布置花坛,但病毒侵染却严重地影响其观赏价值。国内外有关危害矮牵牛的病毒报道较多［１,２］,但较系统研究矮牵牛病毒病的报道较少,本文从矮牵牛病毒鉴定、优势病毒种类确定及防治矮牵牛病毒病进行了初步研究,现报道如下：材料与方法...     

芝麻病毒病病原研究——Ⅰ.芝麻矮化坏死病害的鉴定

在湖北武昌芝麻上分离到的矮化坏死分离物(DNe-I)侵染芝麻引起严重矮化,叶片皱缩坏死。它能够摩擦接种侵染7科14种植物。在苋色藜、千日红上引起局部坏死斑,侵染油菜和百日菊引起系统花叶和黄化。DNe-I能够被桃蚜、花生蚜以非持久性方式进行传播。病毒体外稳定性状:存活期4天,钝化温度60～65℃,稀释限点4×10~(-4)。提纯病毒为线状粒体,长度为770nm。病毒外壳蛋自为单一亚基组成,分子量为30,700±600D。制备抗血清微量沉淀法测定其效价为1:256。在油菜病组织中,观察到风轮状及长直片层叠聚体类型的胞质内含体。在血清学性质上,该分离物与芜菁花叶病毒密切相关,与花生轻斑驳病毒和花生斑驳病毒弱相关,与大豆花叶病毒和西瓜花叶病毒不相关。基于上述性质,DNe-I被鉴定为芜菁花叶病毒。这是国内芜菁花叶病毒自然侵染芝麻的首次报道。     

由高粱花叶病毒和甘蔗花叶病毒引发的浙江甘蔗花叶病害

从浙江省5个地区采集表现花叶症状的甘蔗病叶,用马铃薯Y病毒科简并引物做PCR扩增及测序鉴定.序列分析表明,5个甘蔗样品均含有高粱花叶病毒(SrMV),其中3个样品中还存在甘蔗花叶病毒(SCMV)的复合侵染.序列比较和系统进化树分析表明,浙江甘蔗样品中的SrMV序列彼此很相似,核苷酸同源性大于93%,与已报道的4个美国分离物在CP区域同源性很高,但是3′非编码区的同源性却仅为70%左右.SCMV欧洲和中国玉米分离物及美国、南非和澳大利亚甘蔗分离物分别形成两个远缘群体,浙江甘蔗分离物群体位于两者之间;群体间CP氨基酸序列同源性均大于80%.甘蔗和玉米上的SCMV差异明显,多为无义突变.     

药用植物柴胡病毒病病原物的分子鉴定

利用非序列依赖性扩增(Sequence-independent amplification,SIA)方法对所采柴胡病样进行分子鉴定。序列测定及分析发现,伴有花叶症状的柴胡受到黄瓜花叶病毒(Cucumber mosaic virus,CMV)和蚕豆萎蔫病毒2号(Broad bean wilt virus 2,BBWV2)的复合侵染。为明确柴胡CMV分离物(CMV-SXCH)和柴胡BBWV2分离物(BBWV2-CH)的分类地位,进一步克隆CMV-SXCH的CP、MP和BBWV2-CH的LCP、SCP;序列比对发现,CMV-SXCH与CMV亚组ⅠB中株系XJ2相似性最高,其核苷酸和氨基酸序列同源性分别为99.1%、99.5%;BBWV2-CH与BBWV2中的K株系相似性最高,核苷酸和氨基酸序列同源性分别为95.4%、99.2%。系统进化树分析表明,CMV-SXCH与大多数CMV中国分离物聚类为一簇,同属于CMV亚组ⅠB;BBWV2-CH与BBWV2韩国K株系聚类为一簇,亲缘关系最近。     

我国真菌传线状小麦花叶病毒病病原初步鉴定为小麦黄花叶病毒（WYMV）

由禾谷多粘菌传播的线状小麦花叶病毒有两种,一种是加拿大首先报道的小麦梭条斑花叶病毒（ＷＳＳＭＶ）,另一种是日本报道的小麦黄花叶病毒（ＷＹＭＶ）。这两种病毒粒子形态以及血清学性质非常相似,但核酸序列存在一定差异。经反转录－聚合酶链反应（ＲＴ－ＰＣＲ）和单链构象多态性分析（ＳＳＣＰ）,明确我国广泛发生的禾谷多粘菌传线状小麦花叶病毒都是小麦黄花叶病毒,但供试的８个分离物ＲＮＡ１和ＲＮＡ２序列存在差异,无     

几种葫芦科蔬菜病毒病的鉴定及药剂防治研究

分别在甘肃酒泉、陕西户县及临潼3地区蔬菜大田采集黄瓜、西葫芦、南瓜3种蔬菜上表现病毒病症状的毒原标样135个,采用鉴别寄主的生物学反应和双抗夹心ELISA法鉴定,结果表明引起上述地区黄瓜、西葫芦、南瓜3种蔬菜病毒病的毒原主要是黄瓜花叶病毒。3．95％“病毒必克”可湿性粉剂防治3种蔬菜病毒病的结果表明,300倍、500倍的病毒必克预防效果可达到75％～78％,且治疗效果明显,药效持久。因此,认为病毒必克是目前理想的植物病毒抑制剂。     

我国真菌传线状小麦花叶病毒病病原初步鉴定为小麦黄花叶病毒(WYMV)

由禾谷多粘菌（Ｐｏｌｙｍｙｘａｇｒａｍｉｎｉｓ）传播的线状小麦花叶病毒有两种,一种是加拿大首先报道的小麦梭条斑花叶病毒（ＷＳＳＭＶ）,另一种是日本报道的小麦黄花叶病毒（ＷＹＭＶ）。这两种病毒粒子形态以及血清学性质非常相似,但核酸序列存在一定差异。经反转录聚合酶链反应（ＲＴＰＣＲ）和单链构象多态性分析（ＳＳＣＰ）,明确我国广泛发生的禾谷多粘菌传线状小麦花叶病毒都是小麦黄花叶病毒,但供试的８个分离物ＲＮＡ１和ＲＮＡ２序列存在差异,无一彼此完全相同     

Oligogenic inheritance for resistance to Zucchini yellow mosaic virus in Cucurbita pepo

‘True French’ is an open‐pollinated cultivar of the Zucchini (Courgette) Group of Cucurbita pepo and is susceptible to Zucchini yellow mosaic virus (ZYMV). Using C. moschata‘Menina’ as the source of ZYMV resistance and following six generations of backcrossing, a true‐breeding line nearly isogenic to ‘True French’, designated 381e, was recovered that carried ZYMV resistance, albeit not at as high a level as in ‘Menina’. ‘True French’ and accession 381e were crossed, and their reciprocal F₁, F², and backcross progenies were grown in a chamber and inoculated with a highly virulent, non‐aphid‐transmissible strain of ZYMV. Nearly all F¹ plants and all plants of the backcross to 381e were classified as resistant. Segregation to resistant and susceptible individuals occurred in the backcross to the susceptible parent, in accordance with a 3:5 three‐gene ratio of resistant: susceptible. The F₂ segregated in accordance with a ratio of 45 resistant : 19 susceptible, which would be obtained if there was one major gene for resistance, Zym‐1 (Zym), and two other genes, herein designated Zym‐2 and Zym‐3, both of which for complementary to Zym‐1. The presence of Zym‐1 and either Zym‐2 or Zym‐3 is necessary for resistance to be expressed in young plants, but the presence of all three might be necessary for resistance to continue to be expressed during subsequent development of the plants. Evidently, Zym‐2 and Zym‐3 are ubiquitous in C. moschata but their susceptible alleles are much more common in C. pepo. As the level of resistance of 381e to ZYMV is not as high as that of C. moschata‘Menina’, additional, as yet unidentified, genes must be involved in conferring high resistance to this virus.     

厚皮甜瓜(Cucumis melo var. reliculatus)的快速繁殖

以厚皮甜瓜 (Cucumismelovar.reliculatus)西薄洛托带腋芽茎段为外植体进行离体快速繁殖研究。结果表明 :在MS BA 0 .5～ 1 .0mg/L IAA 0 .1mg/L的培养基上利于诱导形成丛生芽 ,芽的月增殖系数达到 1 1以上 ;在 1 /2MS IAA 0 .5mg/L培养基上并经暗处理 3d最易生根 ,生根率 90 % ;在蛭石 :草炭土 =1 :1 (体积比 )基质中移栽驯化效果好。试管植株定植大田后种性不变 ,生长和结果习性优于种子苗。     

ACC脱氨酶基因转化白兰瓜的初步研究

用带有ACC脱氨酶基因和卡那霉素抗性基因（NptⅡ,作为报告基因）的工程根癌农杆菌转化白兰瓜子叶。通过组织培养,得到具有卡那霉素（Km)抗性的再生小苗,经NptⅡ报告基因的PCR扩增,ACC脱氨酶基因的Southern点杂交以及ACC脱氨酶活性的生理生化检测可知,ACC脱氨酶基因已成功转入白兰瓜子叶再生小苗,而且不同植株的真叶中表现出不同水平的ACC脱氨酶活性。     

Virus-induced changes in the subcellular distribution of glutathione precursors in Cucurbita pepo (L.)

Changes in glutathione contents occur in plants during environmental stress situations, such as pathogen attack, as the formation of reactive oxygen species leads to the activation of the antioxidative defence system. As glutathione is synthesized out of its constituents cysteine, glycine, and glutamate the availability of these components will limit glutathione synthesis in plants especially during stress situations and therefore the ability of the plant to fight oxidative stress. To gain a deeper insight into possible limitations of glutathione synthesis during pathogen attack the present investigations were aimed to study how the subcellular distribution of glutathione precursors correlates with the subcellular distribution of glutathione during virus attack in plants. Selective antibodies against cysteine, glutamate, and glycine were used to study the impact of Zucchini yellow mosaic virus (ZYMV) infection on glutathione precursor contents within different cell compartments of cells from Cucurbita pepo (L.) plants with the transmission electron microscope (TEM). Generally, levels of cysteine and glutamate were found to be strongly decreased in most cell compartments of younger and older leaves including glutathione-producing cell compartments such as plastids and the cytosol. The strongest decrease of cysteine was found in plastids (- 54 %) and mitochondria (- 51 %) of younger leaves and in vacuoles (- 37 %) and plastids (- 29 %) of older leaves. The strongest decrease of glutamate in younger leaves occurred in peroxisomes (- 67 %) and nuclei (- 58 %) and in peroxisomes (- 64 %) and plastids (- 52 %) of the older ones. Glycine levels were found to be strongly decreased (- 63 % in mitochondria and - 53 % in plastids) in most cell compartments of older leaves and strongly increased (about 50 % in plastids and peroxisomes) in all cell compartments of the younger ones. These results indicate that low glycine contents in the older leaves were responsible for low levels of glutathione in these organs during ZYMV infection rather than limited amounts of cysteine or glutamate. Glutathione precursors were virtually absent in cell walls and intercellular spaces and play therefore no important role during ZYMV attack in the apoplast. While glutamate was absent in vacuoles, elevated levels of glycine (up to 30 %) and decreased cysteine contents (up to - 37 %) were observed in vacuoles during ZYMV infection. The impact of the present results on the current knowledge about glutathione synthesis and degradation on the cellular level during ZYMV infection are discussed.     

乙烯利诱导网纹甜瓜主蔓两性花花芽分化的形态和解剖学研究

以网纹甜瓜(Cucumis melo L.var. reticulatus Naud.)品种’西域1号’为试验材料,于幼苗3叶1心期喷施浓度为150 mg·L^-1的乙烯利溶液进行处理,诱导主蔓形成两性花,以清水为对照,分别对处理和对照植株不同时期的主蔓和侧蔓花芽分化过程进行形态和解剖学观察。结果表明：经乙烯利处理后,幼苗植株主蔓花原基持续向两性花分化,最终发育形成两性花。未经处理植株的主蔓花原基在分化早期与两性花发育过程相同,但在雌蕊出现后,不再继续发育,最终发育形成雄花。处理植株主蔓两性花发育过程与侧蔓两性花发育过程相同。     

甜瓜组织培养过程中的染色体数目变异

以甜瓜组织培养过程中产生的不定芽为材料,观察了不同阶段不定芽中染色体数目发生变异的频率,同时比较了在无外加诱变剂的情况下,甜瓜根尖与组培不定芽之间最大分裂相时间的变化。     

Changes in the subcellular distribution of glutathione during virus infection in Cucurbita pepo (L.)

Changes in the subcellular distribution and quantification of glutathione were studied with electron microscopic immunogold cytochemistry in Zucchini yellow mosaic virus (ZYMV)-infected Styrian pumpkin plants (Cucurbita pepo L. ssp. pepo var. styriaca Greb.) two weeks after inoculation. The amount of gold particles bound to glutathione was statistically evaluated for different cell structures, including mitochondria, plastids, nuclei, peroxisomes, and cytosol. In general, ZYMV-infected plants showed higher gold labelling density in intact mesophyll cells of the 5th (older leaves) and the youngest fully developed leaves (younger leaves), and decreased levels of glutathione within root tip cells when compared to the control. In general, within older and younger leaves the highest amount of gold particles was found in mitochondria and the lowest amount in plastids. In ZYMV-infected older leaves, an increase in glutathione was found in peroxisomes (1.7-fold), the cytosol (1.6-fold), mitochondria (1.4-fold), and nuclei (1.2-fold), whereas glutathione levels in plastids did not differ significantly when compared to control cells. In ZYMV-infected younger leaves elevated glutathione contents were found in the cytosol (3-fold), nuclei (2.1-fold), peroxisomes (1.8-fold), and plastids (1.5-fold), whereas mitochondria showed an insignificant decrease in glutathione levels in comparison to the control. In root tip cells of ZYMV-infected plants the amount of gold particles bound to glutathione was decreased in all investigated cell structures by between 0.7- to 0.8-fold. Additionally, total glutathione contents were determined in older and younger leaves using high-performance liquid chromatography (HPLC), which revealed no significant differences between control and ZYMV-infected leaves. The relevance of the results of both methods were compared and are discussed.     

‘弥河银瓜’高效植株再生体系的建立

建立了以薄皮甜瓜品种‘弥河银瓜’子叶为外植体的高效再生体系。取生长5d的甜瓜子叶为外植体,置于MS+2.0mg·L-16-BA+0.5mg·L-1IBA+3.5%蔗糖+0.7%琼脂(pH5.8)的诱导培养基上培养,可获得100%芽分化频率。在不定芽伸长过程中,生长调节剂最佳浓度为0.5mg·L-16-BA+0.1mg·L-1IAA。潮霉素是较合适的筛选抗生素。     

锌锰钛对接种放线菌剂甜瓜幼苗生长及诱导抗病性的影响

以陕甜3号甜瓜幼苗为材料,采用基质育苗、大田栽培法,研究了2种放线菌剂(Act28和康照)育苗接种和育苗移栽双重接种条件下,微量元素锌、锰、钛对甜瓜植株生长及诱导抗病性的影响.结果显示:(1)与单施菌剂相比,在育苗接种Act28并配施微量元素条件下甜瓜植株总鲜重、根鲜重和蔓叶鲜重分别显著增加31.5%、39.5%和31.5%,在双重接种Act28配施微量元素条件下分别显著增加40.5%、17.6%和40.7%.(2)与单纯菌剂接种相比,在育苗接种和双重接种Act28并配施微量元素下甜瓜叶片过氧化物酶活性分别降低4.4%和10.2%,丙二醛含量分别增加10.9%和16.6%;甜瓜叶片多酚氧化酶、过氧化物酶活性及可溶性蛋白含量在育苗接种康照并配施微量元素下分别较育苗接种康照降低2.2%、38.0%和5.0%,丙二醛含量增加17.7%.(3)甜瓜根系多酚氧化酶活性和丙二醛含量在育苗接种Act28配施微量元素处理下较育苗接种分别显著增加30.7%和42.6%,而在Act28双重接种配施微量元素与双重接种处理间无明显变化;甜瓜根系过氧化物酶活性在育苗接种Act28、康照与微量元素配施处理下,分别较2种菌剂育苗接种处理显著降低14.7%、34.3%,苯丙氨酸解氨酶活性分别明显降低14.9%、21.9%.研究表明,菌剂Act28育苗接种或育苗移栽双重接种条件下,锌、锰、钛溶液对甜瓜植株生长具有显著的促进作用,且微量元素效应与接入的菌剂种类及接种方式有关;微量元素溶液对甜瓜防卫酶活性及抗性物质含量等无显著影响或具有一定程度的负效应,从而导致甜瓜诱导抗病性下降.