Variability of some plant species from different origins and their suitability for virus work

Plants of four species obtained from various sources were raised and compared for their suitability as test plants for a number of viruses. The species comprisedChenopodium amaranticolor (local lesion host of red clover mottle virus),Nicotiana glutinosa (local lesion host of tobacco mosaic virus (TMV)),N. rustica (systemic host of TMV, cucumber mosaic virus and tomato spotted wilt virus) andN. tabacum ‘White Burley’ (systemic host of TMV). Some sources proved to be more attractive as test plants or experimental hosts because of, e.g., production of more local lesions per unit of leaf area (C. amaranticolor andN. glutinosa), distinctness of lesions (C. amaranticolor), development of more pronounced systemic symptoms (N. rustica andN. tabacum ‘White Burley’), better growth characteristics (particularlyC. amaranticolor andN. rustica), smoother leaves being easier to rub (N. glutinosa) or more and bigger leaf formation (particularlyN. rustica). The implications for the interpretation of host range studies and the quest for propagation and assays hosts are discussed.     

Exogenous application of melatonin improves plant resistance to virus infection

In this study, melatonin (MEL)-mediated plant resistance to tobacco mosaic virus (TMV) was examined to study local infection in Nicotiana glutinosa and systemic infection in Solanum lycopersicum. Exogenous application of 100 µm MEL increased anti-virus infection activity to 37.4% in virus-infected N. glutinosa plants. The same treatment significantly reduced relative levels of virus RNA analysed by qRT-PCR and virus titres measured by dot-ELISA, and increased the relative expression levels of the PR1 and PR5 genes analysed by qRT-PCR, in virus-infected S. lycopersicum. MEL treatment induced considerable accumulations of salicylic acid (SA) and nitric oxide (NO) but did not significantly affect production of hydrogen peroxide (H₂O₂) in the virus-infected S. lycopersicum plants. Transgenic nahG N. tabacum was used to determine whether MEL-induced TMV resistance was dependent on the SA pathway. The results showed that the relative RNA level of the TMV analysed by qRT-PCR and virus titres analysed by dot-ELISA were not reduced by the MEL treatment in the nahG transgenic N. tabacum seedlings treated twice with 100 µm MEL. The increased relative expression levels of PR1 and PR5 were greatly reduced when cPTIO, an NO scavenger, was included in the MEL treatment. A working model of MEL-mediated plant resistance to TMV is proposed. MEL-mediated plant resistance to viruses provides a new avenue to control plant viral diseases.     

The TIR–NBS but not LRR domains of two novel N-like proteins are functionally competent to induce the elicitor p50-dependent hypersensitive response

The tobacco N protein recognizes the helicase domain (p50) of the Tobacco mosaic virus (TMV) replicase as an elicitor and mediates hypersensitive response (HR). We obtained two cDNA clones encoding novel N-like (NL) proteins NL-C26 and NL-B69 from Nicotiana tabacum cv. Samsun NN. NL-C26 and NL-B69 had a Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR–NBS–LRR) structure and showed 78% and 73% identities to N, respectively. The NL-C26 and NL-B69 genes were also expressed in N. tabacum cv. Samsun nn, which lacks the N gene. Unlike N, NL-C26 and NL-B69, when coexpressed with p50, failed to induce HR on the sites of agroinfiltration in Samsun nn leaves. However, the elicitor-dependent HR in Samsun nn was induced efficiently by chimeric N proteins with the continuous TIR–NBS domains of NL-C26 and NL-B69. On the other hand, the efficiency of HR induction varied significantly among chimeric N proteins with either of the TIR and NBS domains of the NL proteins. In contrast, chimeras carrying the LRR domains of the NL proteins did not induce HR. Thus, the TIR–NBS domains of NL-C26 and NL-B69 could functionally adapt to the LRR domain of N, which may determine the specificity for the elicitor. We speculate that the NL genes are potential HR-inducing resistance genes for undetermined pathogens other than TMV.     

Increased activities of ribonuclease and protease after challenge in tobacco plants with induced systemic resistance

Inoculation of 3-4 lower leaves of tobacco with tobacco mosaic virus (TMV) increased ribonuclease (RNase) and protease activities in inoculated leaves. Little or no increase in the activities were apparent in upper noninoculated leaves prior to challenge. After challenge with TMV or Peronospora tabacina, RNase activities increased more rapidly in the upper leaves of induced plants as compared to those of noninduced plants. Protease activities in the leaves challenged with P. tabacina or TMV also increased after challenge, but little or no differences in the activities were apparent between induced and noninduced plants. The incubation of purified TMV with leaf extracts obtained from induced challenged, noninduced challenged and noninduced unchallenged plants prior to inoculation did not affect the number of local lesions formed on tobacco plants.     

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。     

丁香酚对烟草抗烟草花叶病毒的诱导作用初探

为探讨植物源化合物丁香酚对烟草抗烟草花叶病毒(TMV)的诱导作用,初步研究了丁香酚对接种TMV的烟草叶片中叶绿素和丙二醛(MDA)含量,防御酶苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)和超氧化物岐化酶(SOD)活性,以及病程相关蛋白基因 PR-1 、PR-3 、PR-5 表达的影响。结果表明:丁香酚处理可促进叶绿素的合成,减轻烟草体内MDA的积累,提高PAL、POD和SOD的活性,增强 PR-1 、PR-3 、PR-5 基因的表达。表明丁香酚可提高植物的诱导抗病性,进而减轻TMV对烟草的危害。     

Induction of antiviral resistance and stimulary effect by oligochitosan in tobacco

Oligochitosan was applied by spraying it on tobacco leaves for inhibition of tobacco mosaic virus (TMV). The maximum inhibition of TMV by oligochitosan was observed when inoculation occurred at 24 h after spraying 50 μg ml⁻¹ oligochitosan. The production of H₂O₂ and NO in epidermal tobacco cells induced by oligochitosan was investigated by epidermal strip bioassay and LSCM, using cell permeable fluorophore diaminofluorescein diacetate (DAF-2D) and 2′,7′-dichlorofluorescin diacetate (H₂DCF-DA), respectively. Epidermal tobacco cells treated with oligochitosan resulted in a strong increase of intracellular NO and H₂O₂. Oligochitosan and NO donor sodium nitroprusside (SNP) induced the defense reaction against tobacco mosaic virus (TMV), and increased phenylalanine ammonia-lyase (PAL) activity. Co-treatment of the tobacco cells with oligochitosan and NO scavenger CPTIO blocked the inducing resistance. The results indicated that the defense response induced by oligochitosan was connected with NO pathway.     

Systemic infection of some N-gene-carrying Nicotiana species and cultivars after inoculation with tobacco mosaic virus

Since July 1974Nicotiana tabacum ‘Samsun NN’ plants, inoculated with the common strain of tobacco mosaic virus (TMV), have occasionally been found to develop necrosis on non-inoculated upper leaves 2–7 days after the local necrotic lesions had appeared on the lower leaves. All these plants had been kept in a growth chamber at 17–20°C. Other tobacco species and cultivars carrying theN gene, such asN. glutinosa andN. tabacum ‘Xanthi-nc’, showed the same phenomenon. Substantial amounts of TMV could be recovered from leaves with systemic symptoms. The systemic necrosis somewhat resembled that caused by tobacco rattle virus (TRV). A number of possible causes, such as high concentration of the inoculum, contamination with another strain of TMV or with TRV, change in the genetic composition of the host plants and certain growing conditions (soil, water, pesticides) were investigated. None of these factors could be held fully responsible for the abnormal systemic reaction, although there was evidence that the soil could sometimes play an important rôle.     

利用黄瓜花叶病毒M和Fny株系 的假重组体分析致病关键因子

 用RT-PCR扩增黄瓜花叶病毒M株系（CMV-M）全长基因组cDNA,成功构建CMV-M RNA2和RNA3侵染性克隆后,与CMV-Fny基因组RNA交换得到3个假重组型病毒 （F1M2F3、F1F2M3、F1M2M3）。用F1M2F3、F1F2M3、F1M2M3分别侵染白肋烟,产生坏死环斑、轻微绿斑驳、明脉、黄白化和叶尖线性化等症状。根据假重组型病毒和野生型病毒的表观症状,分析引起各种症状的关键因子,初步判定：CP基因是诱导花叶症状的关键因子,CMV-Fny RNA2是诱导叶尖线性化的关键因子,CMV-M RNA2是诱导叶尖坏死斑关键因子。实时荧光定量PCR结果显示：野生株CMV-M、CMV-Fny和假重组体F1M2F3、F1F2M3、F1M2M3侵染烟草后引起的症状差异与病毒基因组RNA累积没有直接关系。     

Virus-specific expression of systemic acquired resistance in tobacco mosaic virus- and tobacco necrosis virus-infected ‘Samsun NN’ and ‘Samsun’ tobacco

Systemic acquired resistance induced in intact ‘Samsun NN’ tobacco plants by either tobacco mosaic virus (TMV) or tobacco necrosis virus (TNV) was more effective against challenge inoculation with the same than with the other of the two viruses. However, in trimmed ‘Samsun NN’ plants resistance induced by either of these viruses was stronger against TMV than against TNV. In intact ‘Samsun’ tobacco plants TNV induced a systemic resistance against itself identical to the one expressed in ‘Samsun NN’. Moreover, it induced systemic resistance against TMV as based on a decrease in TMV content in challeng-inoculated leaves. These observations indicate that systemic acquired resistance is not limited to combinations in which both inducing and challenging virus give rise to a hypersensitive reaction, and further point to virus-specific factors regulating the extent of resistance expressed.     

Genetic aspects of polyacrylic-acid-induced resistance to tobacco mosaic virus and tobacco necrosis virus in Nicotiana plants

Among the Nicotiana tabacum cultivars and hybrids tested, polyacrylic acid (PAA) only induced resistance to tobacco mosaic virus (TMV) and tobacco necrosis virus (TNV) in the cultivars Xanthi-nc (NN) and Xanthi (nn) respectively. This varietal response to the PAA treatment could be sexually transmitted. The complete genetic analysis of the inheritance of the PAA response is reported for the first time, demonstrating that the response is a dominant character with a Mendelian segregation which occurs independently of either the N gene responsible for resistance to TMV or the PRb-protein genes. Possible relationships between the N gene, the gene for the PAA response and the PRb genes are discussed.     

Viruses of pepper in plastic houses in Crete

During a survey of virus diseases affecting pepper grown in plastic houses in Crete, during 1984–1986, tomato mosaic virus (ToMV) and tobacco mosaic virus (TMV) were detected. The most common virus was TMV, being present in samples of alle pepper cultivars carrying the L₁ resistance gene, while ToMV was isolated only from susceptible pepper cultivars. According to responses ofCapsicum spp. the isolates from 640 samples checked were classified into three pathotypes: P₀, P_1.2 and P_1.2.3. Results of this study show that P_1.2 represents at present the major threat to the Cretan pepper industry.Samenvatting Bij een in de jaren 1984–1986 gehouden inventarisatie van virusziekten in paprika in plastic-foliekassen op Kreta werd zowel het tabaksmozaïekvirus (TMV) als het tomatemozaïekvirus (ToMV) aangetoond. TMV kwam zeer algemeen voor: het werd aangetroffen in alle monsters van de paprikarassen met het resistentiegen L₁. ToMV werd alleen geïsoleerd uit planten van vatbare paprikarassen. Volgens de waargenomen symptomen die de isolaten van 640 monsters opCapsicum spp. vertoonden, konden de isolaten in drie pathotypen, nl. P₁, P_1,2 en P_1,2,3, worden geklassificeerd. Uit het onderzoek is gebleken dat P_1,2 de belangrijkste bedreiging vormt voor de teelt van paprika op Kreta.     

Influence of an m-type thioredoxin in maize on potyviral infection

Expression of many host genes can be altered during virus infection. In a previous study of sugarcane mosaic virus (SCMV) infection in maize (Zea mays), we observed that expression of ZmTrm2, a gene encoding thioredoxin m, was up-regulated at about 10 days post-inoculation (dpi). In this present study we determined that ZmTrm2 silencing in maize by virus-induced gene silencing significantly enhanced systemic SCMV infection. In contrast transient over-expression of ZmTrm2 in maize protoplasts inhibited accumulation of SCMV viral RNA. Furthermore, we found that in inoculated Nicotiana tabacum leaves transient expression of ZmTrm2 inhibited accumulation of the RNA of tobacco vein-banding mosaic virus (TVBMV), a potyvirus infecting dicotyledonous plants. Interestingly in ZmTrm2 transiently expressed N. tabacum leaves, we detected by semi-quantitative RT-PCR a reduced level of the mRNA of class I beta-1, 3-glucanase (GluI), a protein known to have a role in cell wall callose deposition and viral movement. Our data indicate that the maize ZmTrm2 plays an inhibitory role during infection of plants by SCMV and TVBMV.     

A Severe Hellenic CMV Tomato Isolate: Symptom Variability in Tobacco, Characterization and Discrimination of Variants

A severe strain of Cucumber mosaic virus (CMV) originating from an infected tomato plant (Gastouni-Olympia, Greece) was isolated in tobacco (Nicotiana tabacum cv. Xanthi nc), after three serial local lesion passages in Chenopodium quinoa and designated CMV-G. CMV-G induces yellow mosaic (YM) symptoms in tobacco. When CMV-G was passed mechanically through C. quinoa, phenotypic variants inducing YM or green mild mosaic (MM) in tobacco were isolated. Aphid transmission, from different hosts, appears to be an effective approach for separating MM variants of CMV-G from YM variants. In particular, aphid transmission from zucchini proved to be very efficient in selecting for MM variants. In contrast, aphids transmitted only YM variants from tomato plants. Molecular characterization of CMV-G and its progeny resulted in their classification in the CMV subgroup IB, free of satellite RNA, being the first discovery of the subgroup IB in Greece. In the Solanaceae family (tobacco, tomato, pepper) YM variants induced more severe symptoms than the MM variants. YM and MM phenotype was stable in tobacco for all seven passages tried using the obtained YM and MM variants. Cross-protection experiments showed that an isolated MM variant was able to protect tobacco plants against a challenge infection by a YM variant.     

Effects of Susceptibility of Test Plants on Modes of Cucumovirus Transmission by Myzus persicae

In sequential transmission tests of Peanut stunt virus S (PSV-S) and Cucumber mosaic virus V (CMV-V) using Myzus persicae, these viruses behave as a semipersistent virus in Phaseolus vulgaris, but as a nonpersistent virus in Nicotiana tabacum, regardless of the species of plant used as the virus source. In addition, viruliferous aphids retained virus infectivity and transmitted it to P. vulgaris, even after they lost infectivity to N. tabacum. Apparently, the mode of transmission by the aphids differs depending on the plant species used for the assay. After mechanical inoculation with purified PSV-S or CMV-V, P. vulgaris appeared more susceptible to PSV-S than N. tabacum. However, the susceptibility to CMV-V appeared similar in both assay plants. Received 22 May 2000/ Accepted in revised form 14 September 2000     

Aphid vectors of potato virus YN

BesideMyzus persicae a dozen other species were found to be vectors of potato virus Y^N. Eleven other species did not transmit the virus.White Burley tobacco and A6 potato are equally suitable as test plant to monitor the efficency ofRhopalosiphum padi as vector of PVY^N, but as PVY^N source tobacco is not suitable for this aphid species.Between some aphid species rather large differences exist in retention periods of PVY^N. WithR. insertum andAphis fabae transmission after a 1 h starvation period was still 50% of that without starvation. WithPhorodon humuli, M. certus andM. persicae this value was only 15, 30 and 30%, respectively.Samenvatting Van 12 bladluissoorten werd vastgesteld dat zij, evenalsMyzus persicae, vectoren van het aardappelvirus Y^N (PVY^N) zijn. Van 11 andere soorten kon dit niet worden vastgesteld. Nicotiana tabacum cv. White Burley enSolanum tuberosum cv. A6 bleken beide goed bruikbaar als toetsplant voor het vaststellen van de efficiëntie vanRhopalosiphum padi als vector van het PVY^N; voor deze bladluissoort is tabak ongeschikt als bron van PVY^N.De retentieperiode van het PVY^N lijkt bij verschillende bladluissoorten aanzienlijk te variëren. BijRhopalosiphum insertum enAphis fabae bracht één uur vasten na de acquisitie de overbrenging terug tot 50% van die welke zonder vasten werd verkregen. BijPhorodon humuli was de reductie in overbrenging na één uur vasten 85%, bijMyzus certus enM. persicae was deze 70%.