The establishment of a new strain of tobacco mosaic virus resulting from the use of resistant varieties of tomato

Tomato varieties with monogenic resistance (tolerance) to tobacco mosaic virus (TMV) were used by British growers for the first time in 1966. Samples of TMV collected before 1966, and in 1967 and 1968, were tested for their ability to produce virus symptoms on a series of isogenic tomato differential hosts differing in three factors for TMV resistance. Samples collected before 1966 yielded only strain o of TMV, which was unable to overcome any of the three factors. Strain 1, able to infect the newly-introduced resistant varieties, was found with increased frequency after 1966. It was found less frequently on nurseries which had resumed growing susceptible varieties, suggesting that in these it may be unable to compete successfully with strain o. The dangers of indiscriminate release of varieties protected from disease by only one gene are discussed.     

Natural Occurrence of Pepino mosaic virus in Lycopersicon Species in Central and Southern Peru

Pepino mosaic virus (PepMV), a potexvirus first described in 1980 from pepino ( Solanum muricatum ) plants cultivated in Peru, was isolated from diseased tomato plants in the Netherlands in 1999, and is now the cause of an emerging tomato disease in Europe. In a survey of central and southern Peru, 65 wild and four cultivated populations of Lycopersicon , as well as six populations of other species of Solanaceae , were tested for the presence of PepMV and six other viruses. Of the Lycopersicon population sampled, 23 (35.4%) reacted positively in double antibody sandwich (DAS)-enzyme-linked immunosorbent assay (ELISA) with antisera to PepMV. DAS-ELISA tests for PepMV of other solanaceous species were negative, except for one sample of pepino ( Solanum muricatum ). Mechanical inoculation of susceptible Lycopersicon esculentum cv. NE-1 plants with crude sap extracts of 20 of these samples confirmed that 15 of them (from the Departments of Apurimac, Arequipa and Moquegua) were infected with PepMV; these inoculated plants were also DAS-ELISA positive and, in most cases, developed symptoms. Thirteen of the infective extracts were obtained from plants of wild Lycopersicon species (three L. chilense , three L. chmielewskii , two L. parviflorum and five L. peruvianum ) and one each from the cultivated species L. esculentum and S. muricatum . The wild Lycopersicon species are newly reported natural hosts of PepMV. Tests for the other six viruses were negative, except that two samples contained Tomato mosaic virus . Thus, PepMV occurs in Lycopersicon species in central and southern Peru, even in isolated wild populations. These results indicate that the virus is not new to the region and has an efficient mechanism of natural transmission.     

Interaction of cucumber mosaic virus and potato virus y with tobacco mosaic virus

A study was performed on the interaction of cucumber mosaic virus (CMV) of potato virus Y (PVY) with tobacco mosaic virus (TMV). Interference was evaluated using tobacco plantsNicotiana tabacum cv. Java responding to CMV and PVY with a systemic infection and to TMV with local necrotic lesions. The decrease in TMV — induced lesion number gave evidence of a decrease in susceptibility caused by the previous infection with CMV or PVY, the decrease of lesion enlargement demonstrated a decreased TMV reproduction in the plants previously infected with CMV or PVY. The interference concerned was incomplete, as evaluated from reproduction of the challenging TMV and from the decrease in susceptibility of the host to TMV brought about by the first infection with CMV or PVY.     

Resistance to cucumber mosaic virus in potato

Reactions to two subgroup I isolates (Fny-CMV and Pf-CMV) and two subgroup II isolates (A9-CMV and LS-CMV) of cucumber mosaic virus (CMV) were studied in three non tuber-bearing wild potato species (Solanum spp.) of the series Etuberosa, and in two tuber-bearing interspecific potato hybrids and four potato cultivars using graft-inoculation. Three classes of phenotypic reactions (susceptible, hypersensitive, extreme resistance) were observed in the tuber-bearing genotypes. Susceptible genotypes developed mosaic or severe mosaic with leaf malformation and had high CMV titres. Hypersensitive genotypes developed either top necrosis or vein necrosis and/or necrotic spots on apical leaves, and had low CMV titres. Extremely resistant genotypes had no symptoms and no CMV was detected. The hybrid 87HW13.7 (S. tuberosum×S. multidissectum) developed top necrosis specific to infection with Fny-CMV. The hybrid ‘A6’ (S. demissum×S. tuberosum cv. Aquila) was hypersensitive to all CMV isolates tested. Extreme resistance was not functional against all CMV isolates. Neither hypersensitivity nor extreme resistance were related to the CMV subgroup.     

Effects of cucumber mosaic virus infection on electron transport and antioxidant system in chloroplasts and mitochondria of cucumber and tomato leaves

We examined the responses of the photosynthetic and respiratory electron transport and antioxidant systems in cell organelles of cucumber ( Cucumis sativus L.) and tomato ( Lycopersicon esculentum Mill.) leaves to infection of cucumber mosaic virus (CMV) by comparing the gas exchange, Chl fluorescence, respiratory electron transport, superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate–glutathione (AsA–GSH) cycle enzymes and the production of H₂O₂ in chloroplasts, mitochondria and soluble fraction in virus-infected and non-infected leaves. Long-term CMV infection resulted in decreased photosynthesis and respiration rates. Photosynthetic electron flux to carbon reduction, respiratory electron transport via both complex I and complex II and also the Cyt respiration rate all significantly decreased, while photosynthetic alternative electron flux and alternative respiration significantly increased. These changes in electron transport were accompanied by a general increase in the activities of SOD/AsA–GSH cycle enzymes followed by an increased H₂O₂ accumulation in chloroplasts and mitochondria. These results demonstrated that disturbance of photosynthetic and respiratory electron transport by CMV also affected the antioxidative systems, thereby leading to oxidative stress in various organelles.     

Identification of quantitative trait loci associated with resistance to cucumber mosaic virus in Capsicum annuum

QTL analysis for resistance to cucumber mosaic virus (CMV) was performed in an intraspecific Capsicum annuum population. A total of 180 F3 families were derived from a cross between the susceptible bell-type cultivar Maor and the resistant small-fruited Indian line Perennial and inoculated with CMV in three experiments carried out in the USA and Israel using two virus isolates. Mostly RFLP and AFLP markers were used to construct the genetic map, and interval analysis was used for QTL detection. Four QTL were significantly associated with resistance to CMV. Two digenic interactions involving markers with and without an individual effect on CMV resistance were also detected. The QTL controlling the largest percentage (16–33%) of the observed phenotypic variation (cmv11.1) was detected in all three experiments and was also involved in one of the digenic interactions. This QTL is linked to the L locus that confers resistance to tobacco mosaic virus (TMV), confirming earlier anecdotal observations of an association between resistance to CMV and susceptibility to TMV in Perennial. An advanced backcross breeding line from an unrelated population, 3990, selected for resistance to CMV was analyzed for markers covering the genome, allowing the identification of genomic regions introgressed from Perennial. Four of these introgressions included regions associated with QTL for CMV resistance. Markers in two genomic regions that were identified as linked to QTL for CMV resistance were also linked to QTL for fruit weight, confirming additional breeding observations of an association between resistance to CMV originating from Perennial and small fruit weight. Received: 17 July 2000 / Accepted: 16 October 2000     

Inheritance and genetic mapping of cucumber mosaic virus resistance introgressed from Lycopersicon chilense into tomato

Cucumber mosaic virus (CMV) infects a wide variety of crop plants and in tomato (Lycopersicon esculentum Mill.) causes significant economic losses in many growing regions, particularly the Mediterranean. The objective of the present study was to identify the number and map locations of genes controlling resistance to CMV in breeding lines (BC₁–inbreds) derived from the related wild species L. chilense. These lines also carried the gene Tm-2 ^a for resistance to ToMV, which facilitated the interpretation of disease symptoms. The segregation for CMV resistance in the BC₂F₁ and BC₂F₂ generations, following mechanical inoculation with subgroup-I isolates, was consistent with expectations for a single dominant gene, for which the symbol Cmr (cucumber mosaic resistance) was given. Resistant and susceptible BC₁-inbreds were analyzed with RFLP and isozyme markers to identify genomic regions introgressed from L. chilense. The only L. chilense-specific markers found were on chromosome 12; some resistant lines contained a single introgression comprising the entire short arm and part of the long arm of this chromosome, while others contained a recombinant derivative of this introgression. The chromosome 12 markers were significantly associated with CMV resistance in both qualitative and quantitative models of inheritance. The qualitative analysis, however, demonstrated that CMV resistance was not expressed as a reliable monogenic character, suggesting a lack of penetrance, significant environmental effects, or the existence of additional (undetected) resistance factors. In the quantitative analysis, the marker interval TG68 – CT79 showed the most significant association with CMV resistance. No association between CMV resistance and the Tm-2 ^a gene was observed. These breeding lines are potentially useful sources of CMV resistance for tomato improvement, in which context knowledge of the map location of Cmr should accelerate introgression by marker-assisted selection. Received: 9 August 1999 / Accepted: 22 December 1999     

Unique nature of an attenuated strain of tobacco mosaic virus: autoregulation

An attenuated strain L11A of tobacco mosaic virus (TMV) multiplied like wild type strain L at an early stage of infection in tomato leaves. Four days after inoculation, however, multiplication of L11A was drastically reduced (autoregulation) compared with the constant multiplication of L. In mixed infections, L11A strongly inhibited the multiplication of homologous strain L. Experiments with cucumber mosaic virus (CMV) or tobacco plants revealed that the inhibitory mechanism of L11A is not host-specific but virus-specific, and the autoregulatory mechanism is effective only for TMV. RNA synthesis in L11A infected leaves 4 days after inoculation was studied by polyacrylamide gel electrophoresis. Synthesis of TMV-RNA and its replicative intermediate were strongly inhibited, whereas the replicative form of TMV-RNA and ribosomal RNA were synthesized as in the case of L infection. Synthesis of non-coat-protein was studied by the incorporation of radioactive histidine into subcellular fractions derived from leaves infected with L or L11A for 4 days. Different patterns of the two strains in protein synthesis were noted. At least three proteins were predominantly synthesized in L11A infection. One of them was observed in the mitochondria fraction. From its position in polyacrylamide gel, it could be viral coded 165K protein which is considered to be involved in viral RNA replication. These results suggest that the unique nature of attenuated virus L11A, i.e. autoregulation, resulted from the inhibitory mechanism of viral RNA synthesis due to overproduction of 165K protein and is quite distinct from interferon, intrinsic interference or interference by defective virus.     

Co-electroporation of rice protoplasts with RNAs of cucumber mosaic and tobacco mosaic viruses

Cucumber mosaic virus (CMV) RNA was used to study electroporation conditions suitable for protoplasts from rice suspension cultures. Rice protoplasts required a stronger and shorter electric pulse than tobacco protoplasts for introduction of viral RNA. Under optimized conditions, CMV infection was established in 65 % of electroporated protoplasts. In contrast, electroporation with tobacco mosaic virus (TMV) RNA did not result in infection of rice protoplasts. However, when TMV RNA was electroporated into rice protoplasts together with CMV RNA, TMV production was demonstrated in 15 % of protoplasts. Differential staining with fluorescent antibodies against the two viruses showed that the protoplasts producing TMV were without exception also infected by CMV. The results show that CMV replicates in rice protoplasts by itself, whereas TMV does so only with the aid of CMV.Abbreviations CMV cucumber mosaiv virus - PBS phosphate buffered saline - TMV tobacco mosaic virus.     

应用RNAi技术培育抗2种病毒病的转基因烟草

分别提取烟草普通花叶病(TMV)和烟草黄瓜花叶病（CMV）的病毒RNA。经反转录和外壳蛋白阅读框PCR扩增,获得TMV和CMV外壳蛋白基因cDNA, 分别进行两种病毒已知株系cDNA序列比对获得各自的保守序列,设计干涉序列,将干涉片段扩增产物连接到pMD18-T的相邻酶切位点,制备融合序列,并将其正向和反向序列插入pUCCRNAi载体,再转化到pCAMBIA2300-35S-OCS表达载体中。利用农杆菌LBA4404侵染烟草K326,获得3份含有TMV和CMV外壳蛋白基因干涉序列的转化材料,经分子鉴定证实干涉序列已导入烟草,并采用荧光定量PCR技术对其mRNA表达差异进行分析。抗病性调查表明转化烟株对TMV和CMV抗性都显著增强。     

RNA-dependent RNA Polymerase Activities in Tomato Plants Susceptible or Resistant to Tobacco Mosaic Virus

RNA-dependent RNA polymerase activities were measured in healthyand tobacco mosaic virus (TMV)-infected tomato plants, to investigatethe possibility that altered activity might be involved in theoperation of the Tm-I gene for resistance to TMV. Healthy, susceptibleand resistant plants had similar levels of enzyme activity.Infection with TMV strain 0, which is inhibited by Tm-I, causeda 2-fold increase in activity in susceptible plants but no increasein Tm-I plants. Infection with a number of strain 1 isolates,which overcome Tm-I resistance, led to a 2 to 4-fold increasein enzyme activity in resistant plants. RNA-dependent RNA polymerase, Tm-I resistance gene, tobacco mosaic virus, tomato, Lycopersicon esculentum     

Inheritance of resistance to zucchini yellow mosaic virus and watermelon mosaic virus in watermelon

High resistance to zucchini yellow mosaic virus-China strain (ZYMV-CH) and moderate resistance to watermelon mosaic virus (WMV) were found in a selection of PI 595203 (Citrullus lanatus var. lanatus), an Egusi type originally collected in Nigeria. Mixed inoculations showed primarily that these two viruses have no cross-protection. This fact may explain the high frequency of mixed infection often observed in commercial fields. When plants were inoculated with a mixture of the two viruses, the frequency of plants resistant to ZYMV was lower than expected, indicating that WMV infection may reduce the ability of a plant to resist ZYMV. We studied inheritance of resistance to ZYMV-CH and WMV, using crosses between a single-plant selection of PI 595203 and the ZYMV-susceptible watermelon inbreds 9811 and 98R. According to virus ratings of the susceptible parents, the resistant parent, and the F1, F2, and BC1 generations, resistance to ZYMV-CH was conferred by a single recessive gene, for which the symbol zym-CH is suggested. The high tolerance to WMV was controlled by at least two recessive genes.     

Galactolipase activity and free fatty acid levels in chloroplasts of domestic and wild tomatoes with different chilling tolerance

In order to establish differences in the chilling sensitivity of domestic and wild Lycopersicon species, galactolipase (EC 3.1.1.26) activity, free fatty acid (FFA) level and Hill reaction activity were measured in chloroplasts isolated from control and cold treated leaves of L. esculentum Mill., cv. Norton, L. hirsutum Humb. and Bonpl., L. peruvianum var. glandulosum Mill. Galactolipase activity was higher in chloroplasts from Lycopersicon species with high chilling sensitivity than in chloroplasts of more chilling-resistant ones. A similar relationship was observed for FFA level in chloroplasts from both cold-stored and control leaves. Decrease in Hill reaction activity due to cold stress was greater in chloroplasts of more chilling-sensitive species. The changes are accompanied by a decline of photochemical activity. Considering the changes in the three parameters noted above, an increasing order of chilling tolerance was established: L. esculentum < L. hirsutum (700 m) < L. hirsutum (3100 m) < L. peruvianum (3400 m). It is suggested that measurements of galactolipase activity and FFA may be useful in an evaluation of differences in resistance to chilling injury of closely related species.     

QTLs involved in the restriction of cucumber mosaic virus (CMV) long-distance movement in pepper

Partial restriction of cucumber mosaic virus (CMV) long-distance movement originating from the Capsicum annuum inbred line ’Vania’ was assessed in a doubled-haploid progeny using two screening methods: the first allowed one to assess the resistance of adult plants decapitated above the fourth leaf and inoculated on the third leaf using a common CMV strain, and the second allowed one to assess CMV resistance to long-distance movement on seedlings inoculated using an atypical CMV strain. For both resistance tests, the behavior of the F₁ hybrid between ’Vania’ and the susceptible line ’H3’ indicated that partial resistance is inherited as a dominant trait. Phenotypic data from the two screening methods were correlated but the one performed on seedlings was much more severe. A subset of 184 molecular markers well-distributed over the pepper genome was selected for QTL mapping using the composite interval mapping (CIM) method. A total of seven genomic regions, including one major effect and several minor effect QTLs, were shown to be associated with partial restriction of CMV long-distance movement. These results are compared with those already obtained in pepper and also in other solanaceous crops, potato and tomato. Received: 22 March 2001 / Accepted: 9 July 2001     

Extreme resistance to cucumber mosaic virus (CMV) in transgenic tomato expressing one or two viral coat proteins

For the production of broad commercial resistance to cucumber mosaic virus (CMV) infection, tomato plants were transformed with a combination of two coat protein (CP) genes, representing both subgroups of CMV. The CP genes were cloned from the CMV-D strain and Italian CMV isolates (CMV-22 of subgroup I and CMV-PG of subgroup II) which have been shown to produce severe disease symptoms. Four plant transformation vectors were constructed: pMON18774 and pMON18775 (CMV-D CP), pMON18831 (CMV-PG CP) and pMON18833 (CMV-22 CP and CMV-PG CP). Transformed R0 plants were produced and lines were selected based on the combination of three traits: CMV CP expression at the R0 stage, resistance to CMV (subgroup I and/or II) infection in growth chamber tests in R1 expressing plants, and single transgene copy, based on R1 segregation. The results indicate that all four vector constructs generated plants with extremely high resistant to CMV infection. The single and double gene vector construct produced plants with broad resistance against strains of CMV from both subgroups I and II at high frequency. The engineered resistance is of practical value and will be applied for major Italian tomato varieties. This revised version was published online in June 2006 with corrections to the Cover Date.     

Study of multiplication of cucumber mosaic virus in susceptible and resistant Capsicum annuuum lines

A previous survey on pepper lines (Capsicum annuum L.) indicated that a susceptible cultivar, Yolo Wonder, reacted to cucumber mosaic virus (CMV) by producing a systemic yellow mosaic. By contrast, CMV caused no symptoms on lines Perennial and Vania. The virus is recoverable from the uninoculated leaves of Perennial, while in Vania CMV is restricted to the inoculated leaves. To interpret these phenomena, a comparative study on CMV multiplication rates, yield, specific infectivity and relative proportion of RNAs was made in the inoculated leaves of the three pepper varieties. The rate of CMV multiplication, as estimated by the double antibody sandwich form of enzyme-linked immu-nosorbent assay, was lower in Perennial than in Vania or Yolo Wonder. The yield of virus purified from Perennial was very low when compared with Vania or Yolo Wonder. The specific infectivity of the virus extracted from Perennial was less than that from Vania or Yolo Wonder. These results suggest that Perennial is resistant to CMV multiplication, while restriction of the virus in inoculated leaves of Vania is not due to the inhibition of the virus replication. However, polyacrylamide gel electrophoresis revealed that the RNA profiles of CMV purified from the three pepper lines were similar.