引起番茄坏死病的黄瓜花叶病毒TN分离物的研究

从南京郊区田间番茄坏死病株中分离出黄瓜花叶病毒ＴＮ分离物,经人工接种１０科３９种植物,能侵染其中的８科２６种,接种番茄,辣椒和普通烟都引起坏死症状。ＴＮ分离物的失毒温度为５０℃,稀释限点５×１０￣（－３）－５×１０￣（－４）,体外存活期１－２天。蚜虫以非持久性方式传毒,经免疫双扩散测定,ＴＮ分离物与黄瓜花叶病毒抗血清呈阳性反应。用差速离心提纯的病毒粒子经电镜观察为廿面体,直径为２８ｎｍ。ＳＤＳ－ＰＡＧＥ分析病毒外壳蛋白为单一组份,分子量２７５００Ｄ。病毒核酸组份分析发现,该病毒分离物含有ＣＭＶ基因组核酸和一个低分子量的卫星ＲＮＡ。以上结果同已报道的引起番茄坏死的带卫星ＲＮＡ的黄瓜花叶病毒株系一致（Ｋｅａｒｎｅｙ,Ｃ．Ｍ．１９９０;Ｊｏｒｄａ,Ｃ．１９９２）。本文还讨论了该病害的发生和流行。     

受番茄花叶病毒侵染后寄主的超微病变研究

电镜观察了番茄花叶病毒（ＴｏＭＶ）侵染不同寄主的细胞超微结构变化。在２５℃下ＴｏＭＶ侵染番茄（ＬｙｃｏｐｅｒｓｉｃｏｎｅｓｃｕｌｅｎｔｕｍＭｉｌｌ）后,病毒粒子在叶片的表皮,薄壁细胞,维管束组织的细胞质中形成大块结晶体和类结晶体,液泡膜处产生小泡结构,有多泡体和髓鞘样结构构伸入液泡中,在２５℃下ＴｏＭＶ侵染珊西烟（ｉｃｏｔｉａｎａｔａｌａｃｕｍＬ．ｃｖ．Ｘａｎｔｈｉｎｎ）后,除存在病毒结晶体和类结     

蚕豆萎蔫病毒—新疆番茄分离株的鉴定

在新疆番茄斑驳病株上分离出一种球状病毒,回接到番茄上产生斑驳症状。病毒粒体为２０面体,平均直径２５ｎｍ。经汁液摩擦接种可感染昆诺阿藜、苋色藜、蚕豆、番茄等１８种植物,不感染菜豆、豇豆、豌豆、六叶茄、黄瓜等。可由桃蚜传毒。在琼脂双扩散试验中,能与蚕豆萎蔫病毒（ＢＢＷＶ）抗血清产生明显的沉淀线,与豇豆花叶病毒（ＣｐＭＶ）、黄瓜花叶病毒（ＣＭＶ）抗血清均不发生反应。纯化病毒紫外扫描呈典型的核蛋白吸收叫线,病毒衣壳蛋白由两种蛋白亚基构成,其分子量分别为４５９００和２０４００道尔顿,由１８种氨基酸约２５５个氨基酸残基组成。病毒核酸是双组份的,它们的分子量为１３２００００和２３４００００道尔顿。根据上述结果认为该病毒是蚕豆萎蔫病毒侵染番茄的一个株系。     

茄子花叶病新毒原

１９９１年从沈阳地区茄子花叶病叶片中分离到了ＳＹ－Ｉｓ分离物。经汁液摩擦接种７个科１６种植物,这一分离物可浸染４个科８种植物,但不侵染黄瓜、两葫芦和蚕豆,也不能经桃蚜和萝卜蚜传播。该分离物体外抗性的致死温度（ＴＩＰ）为９０－９５℃;稀释限点（ＤＥＰ）为１０￣（－６）－１０￣（－７）;体外保毒期（Ｌ）为一个月以上。光学显微镜下可见晶状胞质内含体。叶滴法电镜观察可见大小为±３００×１８ｎｍ的杆状病毒粒子。该病病叶汁液与烟草花叶病毒（ＴＭＶ）普通株抗血清呈明显阳性反应。用ＴＭＶ的一对引物进行多聚酶链反应（ＰＣＲ）,可扩增出一特异性核酸片段。根据上述实验结果,我们认为引起沈阳地区茄子花叶病的毒原种类为ＴＭＶ,是茄子花叶病的新毒原。     

侵染蚕豆的烟草花叶病毒研究

１９９３年４月从杭州蚕豆田病株上分离获得病毒分离物Ｂ－９３５Ａ,汁液摩擦接种５科１９种植物,Ｂ－９３５Ａ能侵染３科１２种植物。被侵染的蚕豆产生黄斑、花叶,后期叶片黄化并有坏列;在菜豆上产生局部枯斑。Ｂ－９３５Ａ的钝化温度为８５－９０摄氏度,稀释限点为１０＾－８－１０＾－７。病毒粒体杆状,平均长度约３０ｎｍ,病毒的衣壳蛋白亚基只有一条多肽链,分子量约为１７．５ＫＤ,包裹的核酸为单链ＲＮＡ,长约６．４     

侵染新疆甜菜的两种病毒分离物的研究

在新疆甜菜的根部和叶片分离到两个球状病毒分离物。从病毒形态、生物学及物化性质等研究结果证明这两个分离物实是一种病毒。病毒粒体力２０面体,直径约２８ｎｍ,回接到甜菜上产生环斑和沿脉线纹最后形成坏死斑。此外还侵染苋色藜,昆诺阿黎,番杏,菠菜等,引起局部坏死斑。在普通烟,心叶烟,菜豆,黄瓜,蕃茄上无症状侵染,病毒致死温度为７０℃,１０分钟。体外存括期１３天以上,冻干病叶７年后仍有侵染力。通过ＰＥＧ沉淀、差速离心、琼脂糖柱层析及蔗糖梯度离心,可得到较高浓度的纯化病毒,病毒的紫外吸收最低值在２４５ｎｍ,最高值在２６０ｎｍ。经ＳＤＳ－ＰＡＧＥ测定,病毒外壳蛋白的分子量为２．７×１０￣４道尔顿,病毒基因组核酸为三个组份,分子量分别为３．０８ｋｂ,１．２８ｋｂ和０．８５ｋｂ。在琼脂双扩散实验中,能与番茄黑环病毒（ＴＢＲＶ）抗血清产生较弱的沉淀线,与黄瓜花叶病毒（ＣＷＶ）、烟草环斑病毒（ＴＲＳＶ）、烟草坏死病毒（ＴＮＶ）、香石竹环斑病毒（ＣａＲＳＶ）的抗血清不发生反应。     

烟叶脉坏死病毒原鉴定及cDNA克隆与序列分析

田间采集辽宁地区烟叶脉坏死病标样所得分离物,在测定的１２个科３５种植物中只侵染茄科的一些烟草品种及洋酸浆（ｐｈｙｓａｌｉｓｆｌｏｒｉｄａｎａ）,可由桃蚜（Ｍｙｚｕｓｐｅｒｓｉｃａｅ）传播。病叶汁液稀释限点（ＤＥＰ）为１０￣（－２）－v１０￣（－３）;失毒温度（ＴＩＰ）为５５一６０℃;体外保毒期（Ｌ）为４８－７２小时。病毒粒体形态呈线条状７２０×１２ｎｍ,病叶脉坏死部细胞质中含风轮状内含体。病毒提取物的紫外最大吸收为２６５ｎｍ,最小吸收为２４５ｎｍ,Ａ＿（２８０）／Ａ＿（２６０）为０．８２。该病毒分离物与ＰＶＹ￣０抗血清呈阳性反应。以病毒ＲＮＡ为模板,按国外报道的ＰＶＹ￣Ｎ序列合成引物经逆转录合成ｃＤＮＡ。用ＰＣＲ扩增出约０．８０ｋｂ的ＣＰ基因片段,将这一片段插入载体ｐＧＥＭ７Ｚ－ｆ（＋）中转化Ｅ．ｃｏｌｉＤＨ５ａ菌株得到了ＣＰ基因的克隆。ｃＤＮＡ序列分析表明,和国外报道的ＰＶＹ￣Ｎ序列同源性极高,初步表明引起辽宁地区烟叶脉坏死病的毒原为ＰＶＹ￣Ｎ。     

番茄花叶病毒外壳蛋白基因及3′端非编码区的克隆、序列分析及其表达

根据已报道的番茄花叶病毒Ｌ株系（ＴｏＭＶＬ）序列人工合成引物,经ＲＴＰＣＲ扩增并克隆了我国番茄花叶病毒分离物（ＴｏＭＶＳ１）的外壳蛋白ＣＰ基因及３′端非编码区。序列测定结果表明,所得ｃＤＮＡ共长６８２个核苷酸,其中ＣＰ基因含４８０个核苷酸,编码１５８个氨基酸,３′端非编码区含２０２个核苷酸,其核苷酸序列与ＴｏＭＶＬ株系具有９９．５％的同源率。将该基因片段克隆到ｐＧＥＭＥＸ１载体中,转入Ｅ．ｃｏｌｉ后诱导表达,经Ｗｅｓｔｅｒｎｂｌｏｔ检测证明,该基因已在大肠杆菌中正确表达。这是我国首次报道ＴｏＭＶＣＰ基因序列。     

对TMV不同抗性番茄品种叶绿体DNA限制性差异片段的克隆

对ＴＭＶ不同抗性番茄品种ｃｔ－ＤＮＡ经限制性内酶切后,将所获得的差异片段Ｂａｍ６克隆到质粒ｐＢｌｕｅｓｃｒｉｐｔⅡＳＫ中,经筛选、菌落原位杂交、斑点杂交及电泳鉴定,证明重组体为抗性品种Ｂａｍ６差异片段克隆,对探讨番茄ｃｔ－ＤＮＡ与抗ＴＭＶ的关系有重要意义。     

宁夏甜菜的烟草环斑病毒的鉴定

从宁夏患丛根病甜菜的病根中分离到一种球状病毒粒子,直径约为２８ｎｍ。提纯病毒的紫外扫描呈典型的核蛋白曲线,最大吸收为２６０ｎｍ,最小吸收为２４０ｎｍ,Ａ＿（２６０）／Ａ＿（２８０）＝１．３０。寄主范围广,能侵染茄科、豆科、藜科、葫芦科、番杏科等１７种植物。病毒的ＴＤＰ为６５℃,ＤＥＰ为１０￣（－３）,体外存活期６－７天。有较强的免疫原性。病毒的抗血清与烟草环斑病毒（ＴＲＳＶ）之间产生明显的沉淀线。经初步鉴定认为该分离物是烟草环斑病毒。     

番茄多胁迫诱导型LeMTshsp 启动子的分子克隆及其功能分析

根据Southern 杂交结果, 选取KpnⅠ与EcoRⅠ双酶切番茄中蔬4 号基因组DNA, 3 kb 左右的酶切片段连入pBSⅡKS ( + ) 载体, 构建成含有线粒体小分子热激蛋白基因( LeMTshsp) 上游2 kb 左右调控区的质粒文库。通过巢式PCR 方法从构建的质粒文库中克隆出LeMTshsp 基因上游1915 bp 的调控区( GenBank登录号为AB239774) 。该序列含有TATA box 及CAAT box 等启动子基本元件, 还具有6 组典型的HSE 元件及多个AT-rich 区, 另外还有许多逆境反应元件如ABRE , C-repeat— DRE , AP-1。凝胶阻滞结果表明, 纯化的HsfA2 蛋白与LeMTshsp 启动子的HSE 元件在体外具有结合活性, 且与近端5 组HSE 的结合活性比与远端HSE 的结合活性强。构建该启动子与GUS 基因的融合载体, 利用农杆菌介导的叶圆盘法转化番茄, GUS 组织化学染色结果表明LeMTshsp 启动子对热激、低温、外源ABA 及重金属胁迫都有应答     

天津近郊番茄病毒类型的研究

应用ELISA间接法检测出天津近郊番茄上至少有9种病毒类型。且首次检测出国内在番茄上尚未报道的番茄黑环病毒(TBRV)、绒毛烟斑驳病毒(VTMoV)。经生物学测定还分离出番茄上表现枯斑型的86-125和在豇豆上产生大红斑的86-95-15均与13种抗血清呈阴性反应的杆状待测病毒。     

利用多重PCR反应同时筛选番茄Cf-9和Tm-1基因

利用同一PCR反应体系,对分别与番茄抗叶霉病的Cf-9基因和抗番茄烟草花叶病毒病的Tm-1基因紧密连锁的PCR标记进行了同时扩增筛选,扩增的特异性片段与单引物扩增片段吻合。其中与Cf-9基因紧密连锁的CAPs标记在抗感试材均可扩增出560bp的特异片段,且都存在TaqⅠ酶切位点,抗病基因型酶切后分别产生了450bp、330bp和290bp的不同特异性片段,而感病基因型试材酶切后产生450bp和290bp的特异性片段;与Tm-1基因紧密连锁的SCAR标记为显性标记,只有抗病试材产生750bp的特异片段,不能被TaqⅠ酶切。经反复验证,结果稳定准确,可用于在同一PCR反应体系中对两个抗病基因进行同时筛选鉴定。该体系的建立不仅省时、省工、节省费用,而且可用于苗期辅助选育,加快番茄抗病育种进程。     

Oviposition induced volatiles in tomato plants

In response to attack by herbivorous insect, plants produce semiochemicals which act to attract their natural enemies. Recent work on plant volatile compounds has shown that they may play multiple roles as communication signals and defense agents. Here we measured the volatile profile of tomato plants with and without oviposition by the herbivore moth, Tuta absoluta. We used solid phase micro extraction (SPME) sample pre-concentration techniques combined with gas chromatography-mass spectrometry. We have found that different volatile profiles emitted from tomato plants with oviposition by T. absoluta compared to control plants. Hexanal, (Z)-3-hexen-1-ol and an unidentified compound were isolated only from tomato plants with T. absoluta eggs. (Z)-3-hexen-1-ol was identified only from tomato plants with T. absoluta eggs that were laid three days earlier. The percentage of sesquiterpenes (e.g. δ-elemene) has been found to increase in the headspace of tomato plants with oviposition. These results in respect to indirect defense of tomato plants to T. absoluta are discussed.     

Studies on the fungi associated with tomato fruit rots and effects of environment on storage

Seven fungi associated with fruit rot of tomato were isolated includingFusarium equiseti, F. chlamydosporum, Alternaria solani, Geotrichum candidum, Acremonium recifei, Aspergillus flavus andA. niger. They were all pathogenic on tomato fruits, most pathogenic beingGeotrichum candidum followed byA. niger. Least rot was caused byAlternaria solani. The optimum temperature for maximum rotting caused byG. candidum, A. niger andA. flavus was 30°C. The relative humidity for maximum rot ranged from 70–90%. Tomato fruits stored well at 0–10°C and rather poorly at 20–30°C. Fruits stored at 35°C showed blemishes. The best RH for storage ranged between 60 and 90%.     

Evaluation of barrier plants for the cultural control of tomato yellow leaf curl disease

Barrier cropping plays an essential role in controlling insect pests and insect-transmitted diseases in cultural control. It has been proven efficient in suppressing the spread of nonpersistently transmitted viruses. For suppressing the spread of persistently transmitted viruses, barrier cropping is not considered an effective control strategy because barrier plants cannot act as a virus sink to purge the virus in the vector. However, few successful cases of barrier cropping suppressing the spread of persistently transmitted viruses have been reported. The objectives of the present study were to screen candidates (cucumber, okra, Chinese kale, soybean, and corn) for potential barrier plants to control tomato yellow leaf curl Thailand virus (TYLCTHV) and examine whether prefeeding on these plants can reduce the virus titer in its vector, Bemisia tabaci, thus reducing TYLCTHV transmission. The results revealed that nonviruliferous whiteflies preferred cucumber and okra to tomato, whereas viruliferous whiteflies preferred cucumber to tomato. Although prefeeding on cucumber, okra, and Chinese kale did not reduce the titer of TYLCTHV in viruliferous whiteflies, the vector transmission rate decreased after the whiteflies fed on Chinese kale. It implies that planting Chinese kale as a barrier plant for tomato cultivation may reduce the incidence of TYLCTHV. In addition, the preference to cucumber plants may reduce the incidence of whiteflies acquiring TYLCTHV from virus-infected tomato plants and of viruliferous whiteflies inoculating the virus into healthy tomato plants, thereby reducing the disease incidence. Further field trials of barrier cropping using the candidate plants are warranted.     

A transformation procedure for recalcitrant tomato by addressing transgenic plant-recovery limiting factors

Agrobacterium tumefaciens technology is the battle horse for tomato genetic transformation. However, tomato varieties with low regeneration capacity are very difficult to transform. In the past, tomato transformation through Agrobacterium infection was focused on varieties capable of high regeneration yield, while successful transformation of low regenerable cultivars has not been reported. The genotype response to tissue culture conditions is believed to drive the frequency of regeneration of transgenic plant, whereas the capacity for cell proliferation could determine the transformation efficiency through this technology. The Campbell-28 cultivar is an example of constraints arising from a high morphogenetic potential with low conversion compared to normal plants. In the present work the roles that contribute to improved transgenic plant recovery from this recalcitrant variety were explored for factors like Agrobacterium concentration and antibiotics for bacterial removal and transformant selection. Analysis of the efficiency from independent transformation experiments revealed a more than twofold increase of transformant regeneration after selection on ammonium glufosinate compared to kanamycin selection, showing a transformation efficiency of 21.5%.     

番茄凝集素的分离纯化及某些性质的研究

番茄成熟果实经抽提上鸡卵类粘蛋白(OM)-Sepharose 4B亲和层析柱分离纯化制得番茄凝集素。制品在SDS—聚丙烯酰胺凝胶电泳上呈一条带,表观分子量为205,000。Sephadex G200凝胶过滤行为呈单一吸收峰,分子量为180,000。等电聚焦测得等电点为7.6和9.4。它能使人和多种动物红细胞,以及某些培养细胞凝集,但效价不同。N—乙酰葡萄糖胺寡聚糖是其专一性结合糖。     

Biological control of eucalyptus bacterial wilt with rhizobacteria

The antagonistic potential of 298 rhizobacteria obtained from the rhizosphere and rhizoplane of tomato and eucalyptus plants was assessed for the control of bacterial wilt of eucalyptus caused by Ralstonia solanacearum. Several tests were performed using tomato plants as a screening system to select efficient rhizobacteria. Different methods for antagonist delivery and pathogen inoculation were evaluated: (1) seeds were microbiolized (soaked for 12 h in a suspension of the antagonist propagules) and germinated seedlings had their roots immersed in the pathogen inoculum suspension; (2) seedlings originated from microbiolized seeds were transplanted to soil infested with R. solanacearum and (3) roots of seedlings were immersed in a suspension of propagules of the antagonist and subsequently in a suspension of R. solanacearum. Nine isolates (UFV-11, 32, 40, 56, 62, 101, 170, 229, and 270) were selected as potential antagonists to R. solanacearum as they suppressed bacterial wilt in at least one of the methods assessed. The selected antagonists were evaluated against two isolates of R. solanacearum using in vitro and in vivo (inoculated eucalyptus) tests. Isolates UFV-56 (Bacillus thuringiensis), UFV-62 (Bacillus cereus) and a commercial formulation of several rhizobacteria (Rizolyptus®) suppressed bacterial wilt in eucalyptus protecting the plants during the early stages of development.     

营养膜无土栽培技术的研究

试验研究着重于设备、肥料和配方,产量效益、经济成本等。采用控制和调节pH值、电导度、氧气和设备等措施,使黄瓜亩产量达6861.5公斤;番茄因品种不同而有差异,早丰番茄品种达8361.1公斤。番茄每公斤成本0.29元。植物群体高大,根系发达,结果早,个体大,质量好。证明该技术操作容易,管理简单,产量高,有明显的经济效益。