Alfalfa mosaic virus in lucerne seed during seed maturation and storage, and in seedlings

The incidence of alfalfa mosaic virus (AMV) in lucerne seed and pods during maturation, when monitored by sap transmission to Phaseolus (infective virus) and ELISA (AMV antigen), showed that infective virus incidence decreased rapidly with maturation, whereas antigen incidence declined slowly and was always higher than infective virus. Infective virus and antigen incidence were higher in mature seed of cv. Maris Kabul than cv. Europe because virus inactivation/degradation were more rapid in cv. Europe. Seed infection with virus originating from pollen, ovules or both was found in pods and seeds 12–15 days after pollination between healthy or AMV-infected plants; this was before maturation-associated virus inactivation. Ovule transmission was more frequent than pollen transmission. AMV antigen was present in embryos and testas of mature seed; infective virus only in embryos. Non-infective but ELISA-positive antigen in testa extracts accounted for the higher incidence of 'seed-borne AMV' compared with embryo-associated seed transmission to seedlings. Tests with dry mature seed either underestimated (infectivity tests) or overestimated (ELISA) eventual seedling infection. Infectivity and ELISA tests gave identical incidence values for 17 to 29-day-old seedlings.     

Pathogenesis of Alfalfa mosaic virus in Soybean (Glycine max) and Expression of Chimeric Rabies Peptide in Virus-Infected Soybean Plants

ABSTRACT Infection of soybean (Glycine max) plants inoculated with particles of Alfalfa mosaic virus (AlMV) isolate 425 at 12 days after germination was monitored throughout the life cycle of the plant (vegetative growth, flowering, seed formation, and seed maturation) by western blot analysis of tissue samples. At 8 to 10 days after inoculation, the upper uninoculated leaves showed symptoms of virus infection and accumulation of viral coat protein (CP). Virus CP was detectable in leaves, stem, roots, seedpods, and seed coat up to 45 days postinoculation (dpi), but only in the seedpod and seed coat at 65 dpi. No virus accumulation was detected in embryos and cotyledons at any time during infection, and no seed transmission of virus was observed. Soybean plants inoculated with recombinant AlMV passaged from upper uninoculated leaves of infected plants showed accumulation of full-length chimeric AlMV CP containing rabies antigen in systemically infected leaves and seed coat. These results suggest the potential usefulness of plants and plant viruses as vehicles for producing proteins of biomedical importance in a safe and inexpensive manner. Moreover, even the soybean seed coat, treated as waste tissue during conventional processing for oil and other products, may be utilized for the expression of value-added proteins.     

地高辛标记的cDNA探针检测烟草花叶病毒、黄瓜花叶病毒及马铃薯Y病毒

 根据已发表的烟草花叶病毒(Tobacco mosaic virus,TMV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)和马铃薯Y病毒(Potato virus Y,PVY)的外壳蛋白基因序列,设计特异引物,分别以提取的TMV、CMV和PVY侵染的病叶总RNA为模板,反转录PCR进行体外扩增,分别得到长度为0.44、0.77、0.80 kb的目的片段,并克隆到pGEM-T easy质粒载体上,以构建的重组质粒为模板,用PCR方法合成了相应的地高辛标记的双链DNA探针。以合成的探针通过斑点杂交技术检测烟草病叶总RNA和烟草病叶汁液。TMV、CMV和PVY的3种地高辛探针检测各自感染的烟草病叶总RNA的稀释低限分别为1:1000、1:10000、1:320,检测各自侵染烟草病汁液的最大稀释倍数分别为1:100、1:100、1:10,而每种探针与健康烟草和其它2种病毒的反应均为阴性。     

瞬时表达靶向TMV外壳蛋白基因的siRNA能干扰病毒侵染

 RNA干扰(RNA interference,RNAi)是与内源性mRNA编码区某段序列同源的双链RNA导入细胞后,该mRNA发生特异性降解,从而导致该基因表达沉默的现象。小干扰RNA(small interfering RNA,siRNA)作为RNAi途径的重要中介,已被广泛应用于动、植物抗病毒治疗研究。本文以烟草花叶病毒(Tobacco mosaic virus,TMV)外壳蛋白基因为靶位,设计合成表达小干扰RNA的寡核苷酸,亚克隆到植物双元表达载体pBI121中,直接转化根癌农杆菌。通过根癌农杆菌介导的瞬时表达法,研究了同源于TMV外壳蛋白的siRNA对TMV侵染的干扰作用。结果表明,瞬时表达的siRNA能够特异性干扰TMV侵染。含有重组表达载体pBI121/siRNA的根癌农杆菌渗入普通烟植株,在TMV接种后14d其上部叶片没有表现典型的花叶症状。对这些叶片进行Northern杂交试验也没有检测到TMV病毒的RNA积累或仅有很少量的积累。在枯斑寄主心叶烟上,siRNA的瞬时表达可使TMV侵染后的枯斑数明显减少,甚至不产生枯斑。此外,同源于TMV外壳蛋白的siRNA瞬时表达对非同源的黄瓜花叶病毒(Cucumber mosaic virus,CMV)没有抑制作用,表明siRNA的干扰作用具有高度的同源依赖性。     

Kazakh strains of tobacco mosaic virus: two strains with potentially destabilizing amino acid substitutions in the coat protein

Some properties of two Kazakh strains (K1 and K2) of tobacco mosaic virus (TMV) are described. K1 had been isolated by Dr M. Gol'din in 1963, and K2 recently in our laboratory. Both strains were rather similar in host range and antigenic properties to the tomato strain of TMV (tomato mosaic virus, ToMV), but differed from the latter by inducing unusual symptoms on upper noninoculated leaves of infected tobacco plants. K1 was semi-defective and temperature-sensitive, and formed large amounts of long RNA-free helical protein rods in infected plants. K2 was found to be neither defective nor temperature-sensitive, and did not produce protein rods in infected cells. K1 and K2 coat protein gene sequencing data showed, as expected, that both proteins are similar in primary structure to ToMV coat protein: only three amino acid substitutions, relative to ToMV, were found in K1 and five in K2 coat protein. Two of these substitutions are unusual, namely, substitution of normally strictly conserved R92 by S (with concomitant Q99→R change) in K2 and substitution of K53 by E in K1.     

<Emphasis Type="Italic">Tobacco mosaic virus</Emphasis> Is Transmissible from Tomato to Tomato by Pollinating Bumblebees

Tobacco mosaic virus (TMV) was detected by ELISA, electron microscopy and/or bioassay from bumblebee (Bumbus terrestris), pollen clumps, nest materials and bee-visited anthers of flowers from greenhouses in which tomatoes had been pollinated by bees and were severely infected with TMV. Experimental bee-mediated transmission of TMV in greenhouse tomatoes demonstrated that the bumblebees transported TMV from plant to plant and that they spread the virus in greenhouses. This is the first report describing TMV transmission by bumblebees. Received 11 August 1999/ Accepted in revised form 30 September 1999     

Occurrence of squash mosaic virus in melons in Greece

Squash mosaic virus (SqMV) was isolated from melon plants raised from imported seed. Virus identification was based on host range, aphid and seed transmission tests, electron microscopy and serology. Seed testing by ELISA detected SqMV only in the cultivar Touralia imported from the USA.     

Variation in the pathogenicity of two Turnip mosaic virus isolates in wild UK Brassica rapa provenances

Brassica rapa can be infected with Turnip mosaic virus (TuMV) as a result of manual inoculation or aphid transmission, but infected plants have not been found in the field. In this study, B. rapa plants grown from seed collected from two field sites in southern England were mechanically inoculated with one of two distinct isolates (pathotypes) of TuMV under glasshouse conditions. These had either been isolated from Brassica oleracea growing wild in Wales, (GBR 83, pathotype 3) or Dorset (GBR 98, pathotype 1). Use of ELISA as an index of infection in manually inoculated B. rapa showed that although seed provenance had a small effect on the proportion of plants infected, the biggest factor was the virus isolate. Both virus isolates infected both lines of B. rapa , but invaded at different rates, although both resulted in easily discernible symptoms. The severity of symptoms was not related to amounts of virus in the infected plants. A significantly greater proportion of plants were infected with GBR 83 at 45 days post-inoculation (d.p.i.) than GBR 98. but GBR 98 caused significantly more severe and obvious symptoms as well as greater mortality at 119 d.p.i., in plants from both sites than GBR 83.     

嘧肽霉素抗烟草花叶病毒作用机理初步研究

 以烟草花叶病毒(TMV)/烟草为测试体系,用³H-尿苷和³H-亮氨酸2种同位素示踪的方法研究了4%嘧肽霉素水剂抗TMV的作用机理。通过³H-尿苷示踪实验发现:无论是叶盘法还是整株法测定,嘧肽霉素均能抑制病毒对³H-尿苷的利用,抑制率分别为72.43%和68.95%;而对寄主植物RNA合成,2种方法的抑制率分别为8.09%和11.02%。通过³H-亮氨酸示踪结果可以看出:嘧肽霉素较强地抑制了病毒对³H-亮氨酸的吸收,但能促进寄主对³H-亮氨酸的吸收。³H-尿苷和³H-亮氨酸分别是RNA和蛋白质合成的前体物,因此,嘧肽霉素通过抑制病毒RNA复制和蛋白质的合成来扰乱病毒的增殖,从而减弱病害的发生。     

<Emphasis Type="Italic">Turnip yellow mosaic virus</Emphasis> isolated from Chinese cabbage in Japan

A virus that caused a distinct yellow mosaic was isolated in Okayama, Japan from Chinese cabbage (Brassica rapa L., Pekinensis group). The virus, with spherical particles ca. 28 nm in diameter, was mechanically transmissible only to cruciferous species. From the host range, characteristic morphology of virus particles, serology and sequence analysis of coat protein gene, the causal virus was identified as Turnip yellow mosaic virus (TYMV). Seed transmission of TYMV at 0–2.2% in Chinese cabbage was confirmed. This report is the first of TYMV from Chinese cabbage and in Japan. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases as accessions AB358971 and AB358972.     

甘蔗花叶病毒在玉米种子中的分布及其与种子传毒的关系

 采用免疫学、电镜观察、RT-PCR、组织培养及生物学测定等方法,针对引起玉米矮花叶病的甘蔗花叶病毒(Sugarcane mosaic virus,SCMV)在高感自交系Mo17的乳熟期、蜡熟期、成熟期和室温贮存的成熟干种子上的分布部位及其各部位病毒的侵染性进行了研究。结果表明:病毒存在于种皮、胚乳的糊粉层和胚内,糊粉层和胚内的病毒具有侵染活力,胚内的可侵染性病毒可以通过发芽传递给下一代幼苗,完成病毒的种子传播过程。在种皮内没有检测到具有侵染活力的病毒,在胚乳的淀粉层内未检测到病毒。在种子成熟过程中,种子内的病毒不断得到积累,随着种子的脱水、干燥又被部分钝化,使得种子传毒率降低。     

进境番茄种子中番茄花叶病毒的检测与鉴定

利用双抗夹心 酶联免疫吸附试验(DAS-ELISA)和反转录 聚合酶链式反应（RT-PCR）方法对来自韩国的番茄种子进行种传病毒检测。结果表明,在该批番茄种子中检测到番茄花叶病毒（ToMV）和烟草花叶病毒（TMV）。PCR产物测序结果表明,ToMV特异引物（ToA/ToB）与TMV特异引物（TA/TB）扩增的片段均为ToMV的外壳蛋白（CP）基因及3′非编码区（3′ UTR）,该片段与其他ToMV分离物的核苷酸序列相似性为98.2%~99.9%。本研究利用重新设计合成TMV和ToMV特异引物对该批种子进行RT PCR检测,仅ToMV特异性引物（ToMf1/ToMr1）扩增到670 bp的预期片段,TMV特异引物（TMf1/TMr1）则未出现特异性扩增,表明重新设计的引物可准确区分ToMV和TMV。以上结果证实该批番茄种子仅携带ToMV。     

青蒿中番茄斑萎病毒和烟草花叶病毒的分子鉴定及相关序列分析

番茄斑萎病毒(tomato spotted wilt virus, TSWV)和烟草花叶病毒(tobacco mosaic virus, TMV)是2种重要的植物病原病毒, 对多种经济作物的产量和品质均造成严重影响。2021年-2022年, 在云南省丽江市烟草种植区不同烟区采集叶片黄化、皱缩以及无症状的青蒿Artemisia caruifolia样品共计14份, 利用免疫金标速测卡和RT-PCR对其病原病毒进行检测。利用免疫金标速测卡检测结果显示, 在所检样品中有9份样品检测出TSWV, 检出率为64.28%, 有3份样品检测出TMV, 检出率为21.43%, 2种病毒复合侵染的检出率同样为21.43%;利用RT-PCR对复合侵染的3份样品进行分子检测, 结果显示, 在3份复合侵染青蒿样品中获得3条TSWV N基因序列、3条TMV cp基因序列和2条TMV RdRp部分序列。TSWV青蒿分离物与分离自云南的TSWV-2分离物相似性最高, 为99.6%;TMV青蒿分离物与分离自辽宁的TMV-Shenyang分离物和分离自云南的TMV-Yongren-1相似性最高, 均大于99.4%。这是首次发现TSWV和TMV 2种不同属病毒复合侵染青蒿。     

侵染扶桑的烟草花叶病毒分离物鉴定

从表现叶斑驳症状的扶桑病株上获得一病毒分离物,电镜下可见约300 nm×18 nm的杆状粒子,其与烟草花叶病毒抗血清呈明显的阳性反应,dsRNA约为6.4 kbp。根据烟草花叶病毒(tobacco.mosaic virus,TMV)的RNA序列设计引物,进行RT-PCR检测,扩增出约800 bp的预期特异片段。将PCR产物连接pMD18-T载体,转化大肠杆菌DH5α,得到了含有目的片段的重组子。序列分析表明,与周雪平等报道的序列(GenBank AJ011933.1)同源性达99％。通过生物学、病毒粒子观察、血清学以及分子生物学实验结果,确定该病毒分离物为TMV。     

Lettuce mosaic virus

Lettuce mosaic virus (LMV) is an economically significant virus of lettuce and endive. The virus has spread world-wide due to the exchange of seed of lettuce varieties. A very high proportion of infected plants may result from a low level of infected seed, because of very efficient transmission by a number of aphid species. The symptoms are characteristic but the diagnosis can be difficult, particularly on lettuce, because numerous viruses may coinfect this species. A very reliable and sensitive method by ELISA has been established for diagnosis and detection, which gives a good estimation ofthe contamination level in a seed batch. The use of virus-free seed, preventive cultural practices and the use of tolerant varieties were shown to be good methods for control if rigorously applied. Up to now, strains able to overcome the genes g and mo, considered to be identical, were shown to be non-seed-transmissible. Studies carried out with several virulent isolates have shown that genes mo and g are different and probably allelic, and that one strain infects seed at a very high level on susceptible and tolerant genotypes. These features have necessitated the production of virus-free seed, including systematic checks on all cultivars, and have stimulated research on new sources of resistance. Recent molecular studies have provided clones for detection and strain differentiation. Assays to introduce different LMV genes into lettuce seem promising.     

五种烟草病毒TMV、CMV、TEV、PVY及TVBMV的多重RT-PCR同步检测

 我国烟草病毒主要有烟草花叶病毒(TMV)、黄瓜花叶病毒(CMV)、烟草蚀纹病毒(TEV)、马铃薯Y病毒(PVY)和烟草脉带花叶病毒(TVBMV),通常发生复合侵染。本研究对我国5种烟草病毒的外壳蛋白基因部分序列设计引物,通过优化引物和模板浓度,摸索扩增参数,在一个体系中成功对5种病毒复合侵染的烟草材料进行多重RT-PCR扩增,得到237、273、347、456和547 bp共5条特异性条带,建立了能同时检测TMV、CMV、TEV、PVY和TVBMV的多重RT-PCR检测体系。对田间样品检测结果证明,多重RT-PCR体系能够同时检测5种病毒,并且灵敏度高。