The prevalence and spatial distribution of viruses in natural populations of Brassica oleracea

We report a survey of four viruses (beet western yellows luteovirus (BWYV), cauliflower mosaic caulimovirus (CaMV), turnip mosaic potyvirus (TuMV), turnip yellow mosaic tymovirus (TYMV)) in five natural populations of Brassica oleracea in Dorset (UK). All four viruses were common; 43% of plants were infected with BWYV, 60% with CaMV, 43% with TuMV and 18% with TYMV. For each virus there were significant differences in the proportion of infected plants among populations, which were not completely explained by differences in the age of plants. Multiple virus infections were prevalent, with 54% of plants having two or more virus types. There were statistically significant associations between pairs of viruses. The CaMV was positively associated with the other three viruses, and BWYV was also positively associated with TuMV. There was no detectable association between BWYV and TYMV, whereas TuMV and TYMV were negatively associated. We suggest these associations result from BWYV, CaMV and TuMV having aphid vectors in common, as aphids are attracted to plants that already have a virus infection. Infected plants were distributed randomly or were very weakly aggregated within populations. The implications of widespread multiple virus infections in natural plant populations are discussed with respect to the release of transgenic plants expressing virus-derived genes.     

Resistance of transformed and non-transformed oilseed rape cv. HM-81 to the infection with cauliflower mosaic,turnip yellow mosaic and turnip mosaic viruses

Resistance of transformed and non-transformed spring oilseed rape cv. HM-81 to the infection with cauliflower mosaic virus (CaMV), turnip yellow mosaic virus (TYMV) and turnip mosaic virus (TuMV) was studied, to determine the influence of transformation on susceptibility of plants to viruses. For experiments the non-segregating R 1 generation of primary transformant HM-81-JZ and control plants of cv. HM-81 were used. The primary transformant was obtained by inoculation of stems withAgrobacterium rhizogenes 15834. All transformed plants of R 1 generation had typically „transformed“ phenotype. No significant differences were revealed in the resistance of both transformed and non-transformed plants to each virus, as proved by qualitative and quantitative ELISA and visual evaluation of symptoms. Transformed plants infected with turnip yellow mosaic virus showed significantly lower reduction of green mass yield than non-transformed. In the case of CaMV and TuMV infection reduction of yield of transformed and non-transformed plants was almost the same.     

Seed transmission of turnip yellow mosaic virus in winter turnip and winter oilseed rapes

This is the first record of seed transmission of turnip yellow mosaic virus (TYMV) in oilseed and turnip rapes. The seed transmission of TYMV in a naturally infected winter turnip rape (Brassica napus var. silvestris) cultivar Perko PVH was investigated. By ELISA 1.6%, 3.2% and 8.3% seed transmission of the virus was found in seed of plants from three localities. The proportion of infected seeds produced by artificially infected plants of winter oilseed rape (Brassica napus ssp. oleifera) and winter turnip rape cultivars was determined. The virus transmission rate, expressed as the proportion of virus-infected plants which germinated from the seed was for the oilseed rape cvs Jet Neuf 0.1%, Solida 0.4%, Silesia 0.8%, Darmor 1.2%, SL-507 0.2%, SL-509 0.0% and for the winter turnip rape cv. Perko 1.5%. ELISA cannot be used in direct tests on bulk seed lots to estimate proportion of infected seed, but must be used on germinated seedlings.     

Characterization of Turnip mosaic virus from the Asian‐BR Population in Iran

Brassicaceae crops in eight provinces of the North‐west Iran were surveyed for Turnip mosaic virus (TuMV) infection during 2011 and 2012. Many symptomatic plants (38%; 226 of 598) were found to be infected with TuMV. The highest frequency was in turnip (61%), followed by radish (55%), oilseed rape (38%), and brassica weeds including annual bastard cabbage (42%), small tumbleweed‐mustard (50%) and wild radish (45%), but not Brassica oleracea and Lepidium sativum. Using biological assays, Iranian TuMV isolates grouped in three [B], [B(R)] and [BR] host‐infecting types. Phylogenetic analysis using complete coat protein (CP) gene nucleotide sequences showed that the Iranian isolates belonged to the Basal‐B and Asian‐BR populations. No evidence of recombination was found in these isolates using different recombination‐detecting programmes. To our knowledge, our study shows for the first time the occurrence of TuMV Asian‐BR subpopulation in the mid Eurasian region of Iran. The data suggest that the Asian‐BR subtype population is found across southern Eurasia and might be a continuous population in East Asia (mostly Japan and China) and Minor Asia (Turkey), the places considered to be one of the origins of TuMV populations.     

Viruses infecting winter oilseed rape (Brassica napus ssp. oleifera)

Turnip mosaic virus (TuMV) and cauliflower mosaic virus (CaMV) have been found infecting field crops of winter oilseed rape (Brassica napus ssp. oleifera) in South Warwickshire. Other viruses found include broccoli necrotic yellows virus (BNYV) and a member of the beet western yellows virus group. Systemic leaf symptoms caused by TuMV varied within and between cultivars; the three predominant reaction types were classified as necrotic, mosaic and immune. Some recently introduced cultivars of oilseed rape were more severely affected by TuMV infection than older cultivars. Reactions to CaMV were less varied and immunity was not found. The seed yield from TuMV and CaMV-infected plants was less than that of healthy control plants. This effect was due to infected plants producing either fewer seeds, smaller seeds or both. Germination of seeds from infected plants was unaffected if sown soon after harvest. After storage for one year the germination of seed from a virus infected plant was significantly less than that of seed from a virus-free plant. All commercial cultivars tested were experimentally susceptible to turnip yellow mosaic virus (TYMV) and some American strains of cucumber mosaic virus (CMV).     

Inhibition of turnip yellow mosaic virus synthesis by pyrimidine analogues

The pyrimidine analogues 2-thiouracil, 2-thiouridine, 6-azauracial and 6-azauridine all inhibited the synthesis of turnip yellow mosaic virus (TYMV) and increased the synthesis of empty virus protein shells in infected Chinese cabbage leaf discs. Uracil and uridine reversed these effects. 2-Thiouracil also reduced the UTP pool in TYMV infected leaf discs. The results are consistent with the suggestion that these analogues or their in vivo derivatives affect virus synthesis by inhibiting the biosynthesis of uridylic acid, possibly by inhibiting orotidylic acid decarboxylase.     

The cis-expression of the coat protein of turnip mosaic virus is essential for viral intercellular movement in plants

To establish infection, plant viruses are evolutionarily empowered with the ability to spread intercellularly. Potyviruses represent the largest group of known plant-infecting RNA viruses, including many agriculturally important viruses. To better understand intercellular movement of potyviruses, we used turnip mosaic virus (TuMV) as a model and constructed a double-fluorescent (green and mCherry) protein-tagged TuMV infectious clone, which allows distinct observation of primary and secondary infected cells. We conducted a series of deletion and mutation analyses to characterize the role of TuMV coat protein (CP) in viral intercellular movement. TuMV CP has 288 amino acids and is composed of three domains: the N-terminus (amino acids 1–97), the core (amino acids 98–245), and the C-terminus (amino acids 246–288). We found that deletion of CP or its segments amino acids 51–199, amino acids 200–283, or amino acids 265–274 abolished the ability of TuMV to spread intercellularly but did not affect virus replication. Interestingly, deletion of amino acids 6–50 in the N-terminus domain resulted in the formation of aberrant virions but did not significantly compromise TuMV cell-to-cell and systemic movement. We identified the charged residues R178 and D222 within the core domain that are essential for virion formation and TuMV local and systemic transport in plants. Moreover, we found that trans-expression of the wild-type CP either by TuMV or through genetic transformation-based stable expression could not rescue the movement defect of CP mutants. Taken together these results suggest that TuMV CP is not essential for viral genome replication but is indispensable for viral intercellular transport where only the cis-expressed CP is functional.     

The incidence of viruses in wild Brassica nigra in Dorset (UK)

Using enzyme‐linked immunosorbent assays, the frequency of occurrence of six viruses was determined in Brassica nigra collected from five coastal sites in Dorset, spanning approximately 24 km. During 1998–2000, the viruses detected were: Turnip mosaic virus (genus Potyvirus) (TuMV), Turnip yellow mosaic virus (genus Tymovirus) (TYMV), Turnip crinkle virus (genus Carmovirus) (TCV), Turnip rosette virus (genus Sobemovirus) (TRoV), Beet western yellows virus (genus Polerovirus) (BWYV) and Cauliflower mosaic virus (genus Caulimovirus) (CaMV). Multiple infections were detected in some individuals (48/447). TuMV was detected infrequently over the three‐year period (5/597). A representative isolate of each virus was tested for its effects on glasshouse‐grown individuals from different half‐sib families of B. nigra from four of the sites. Whether inoculated manually or via aphids (Myzus persicae), TuMV caused a rapid (within 10 days) lethal systemic necrosis in the B. nigra seedlings except when they were near flowering at the time of inoculation. Each of the other viruses invaded systemically but were not lethal. Indeed, BWYV systemically invaded 13/19 glasshouse‐grown B. nigra seedlings but did not produce any visible symptoms. Otherwise, the isolates tested differed in their pathogenicity and in the symptoms they produced in infected B. nigra. With TYMV or TCV viral antigen concentration was closely linked to pathogenicity; for TRoV or CaMV, there was little or no difference in virus concentration between plants with and without symptoms. Substantial and reproducible differences were observed in sensitivity/susceptibility among B. nigra genotypes from different sites in Dorset challenged with the same virus isolate.     

芜菁花叶病毒分子生物学研究进展

芜菁花叶病毒(Turnip mosaic virus,TuMV)是马铃薯Y病毒属(Potyvirus)的一个重要成员, 在世界上分布广泛,主要危害十字花科植物。本文对芜菁花叶病毒的分子生物学相关研究,如基因组结构和功能、株系变异与进化、寄主植物抗性以及抗病毒转基因工程等的研究进展作一阐述。     

Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance

Plant microRNAs (miRNAs) regulate the abundance of target mRNAs by guiding their cleavage at the sequence complementary region. We have modified an Arabidopsis thaliana miR159 precursor to express artificial miRNAs (amiRNAs) targeting viral mRNA sequences encoding two gene silencing suppressors, P69 of turnip yellow mosaic virus (TYMV) and HC-Pro of turnip mosaic virus (TuMV). Production of these amiRNAs requires A. thaliana DICER-like protein 1. Transgenic A. thaliana plants expressing amiR-P69(159) and amiR-HC-Pro(159) are specifically resistant to TYMV and TuMV, respectively. Expression of amiR-TuCP(159) targeting TuMV coat protein sequences also confers specific TuMV resistance. However, transgenic plants that express both amiR-P69(159) and amiR-HC-Pro(159) from a dimeric pre-amiR-P69(159)/amiR-HC-Pro(159) transgene are resistant to both viruses. The virus resistance trait is displayed at the cell level and is hereditable. More important, the resistance trait is maintained at 15 degrees C, a temperature that compromises small interfering RNA-mediated gene silencing. The amiRNA-mediated approach should have broad applicability for engineering multiple virus resistance in crop plants.     

The incidence of viruses in wild Brassica rapa ssp. sylvestris in southern England

Using enzyme‐linked immunosorbent assays, the frequency of occurrence of six viruses was determined in Brassica rapa ssp. sylvestris collected from two Thameside sites (Abingdon and Culham) in Oxfordshire and one near the Avon (Claverton) in Bath & North East Somerset. During 2000–2001, the viruses detected were: Beet western yellows virus (genus Polerovirus) (BWYV), Cauliflower mosaic virus (genus Caulimovirus) (CaMV), Turnip crinkle virus (genus Carmovirus) (TCV), Turnip rosette virus (genus Sobemovirus) (TRoV), and Turnip yellow mosaic virus (genus Tymovirus) (TYMV). BWYV and TYMV were the most frequently detected viruses at the Oxford shire sites, both as single infections (20/1743 and 66/1743 respectively) and as dual infections (7/1743). Turnip mosaic virus (genus Potyvirus) (TuMV) was not detected in the field‐grown plants assayed from any of the sites. There was a highly significant (x²_[1]=30.07, P<0.001) difference in the proportion of plants at each Oxfordshire site in which one or more viruses were detected, and essentially the same pattern of virus infection was observed in tests on B. rapa from the site near Claverton. At least one representative isolate of each detected virus was tested for its morphological and serological effects on glasshouse‐grown individuals from different half‐sib families of B. rapa from both Oxfordshire sites. Except for TRoV, where there was a large difference in the frequency of successful infection in B. rapa from the two locations (1/15 vs 11/15), no clear evidence of resistance or immunity to challenge was observed, although tolerance (virus invasion without symptoms) was frequent. Fewer of the plants from Abingdon were infected than those from Culham, when mechanically challenged with TRoV, but the two B. rapa populations were not otherwise consistently different, either in their infectibility by this virus or in their responses to challenge. However, with TCV, viral antigen concentration was closely linked to the severity of disease and the B. rapa from both Oxfordshire sites segregated into two classes: those with symptoms and most viral antigen, and those without symptoms and least viral antigen. These results suggest that generic risk assessments cannot be made due to differences in the way distinct B. rapa populations react to virus challenge.     

NbALD1 mediates resistance to turnip mosaic virus by regulating the accumulation of salicylic acid and the ethylene pathway in Nicotiana benthamiana

AGD2-LIKE DEFENCE RESPONSE PROTEIN 1 (ALD1) triggers plant defence against bacterial and fungal pathogens by regulating the salicylic acid (SA) pathway and an unknown SA-independent pathway. We now show that Nicotiana benthamiana ALD1 is involved in defence against a virus and that the ethylene pathway also participates in ALD1-mediated resistance. NbALD1 was up-regulated in plants infected with turnip mosaic virus (TuMV). Silencing of NbALD1 facilitated TuMV infection, while overexpression of NbALD1 or exogenous application of pipecolic acid (Pip), the downstream product of ALD1, enhanced resistance to TuMV. The SA content was lower in NbALD1-silenced plants and higher where NbALD1 was overexpressed or following Pip treatments. SA mediated resistance to TuMV and was required for NbALD1-mediated resistance. However, on NahG plants (in which SA cannot accumulate), Pip treatment still alleviated susceptibility to TuMV, further demonstrating the presence of an SA-independent resistance pathway. The ethylene precursor, 1-aminocyclopropanecarboxylic acid (ACC), accumulated in NbALD1-silenced plants but was reduced in plants overexpressing NbALD1 or treated with Pip. Silencing of ACS1, a key gene in the ethylene pathway, alleviated the susceptibility of NbALD1-silenced plants to TuMV, while exogenous application of ACC compromised the resistance of Pip-treated or NbALD1 transgenic plants. The results indicate that NbALD1 mediates resistance to TuMV by positively regulating the resistant SA pathway and negatively regulating the susceptible ethylene pathway.     

The apparent non‐host resistance of Ethiopian mustard to a radish‐infecting strain of Turnip mosaic virus is largely determined by the C‐terminal region of the P3 viral protein

Two different isolates of Turnip mosaic virus (TuMV: UK 1 and JPN 1) belonging to different virus strains were tested on three different Brassica species, namely turnip (Brassica rapa L.), Indian mustard (Brassica juncea L.) and Ethiopian mustard (Brassica carinata A. Braun). Although all three hosts were readily infected by isolate UK 1, isolate JPN 1 was able to establish a visible systemic infection only in the first two. Ethiopian mustard plants showed no local or systemic symptoms, and no virus antigens could be detected by enzyme‐linked immunosorbent assay (ELISA). Thus, this species looks like a non‐host for JPN 1, an apparent situation of non‐host resistance (NHR). Through an experimental approach involving chimeric viruses made by gene interchange between two infectious clones of both virus isolates, the genomic region encoding the C‐terminal domain of viral protein P3 was found to bear the resistance determinant, excluding any involvement of the viral fusion proteins P3N‐PIPO and P3N‐ALT in the resistance. A further determinant refinement identified two adjacent positions (1099 and 1100 of the viral polyprotein) as the main determinants of resistance. Green fluorescent protein (GFP)‐tagged viruses showed that the resistance of Ethiopian mustard to isolate JPN 1 is only apparent, as virus‐induced fluorescence could be found in discrete areas of both inoculated and non‐inoculated leaves. In comparison with other plant–virus combinations of extreme resistance, we propose that Ethiopian mustard shows an apparent NHR to TuMV JPN 1, but not complete immunity or extreme resistance.     

The reactions of some Brussels sprout F1 hybrids and inbreds to cauliflower mosaic and turnip mosaic viruses

Virus infection of Brussels sprout crops was widespread in Britain in 1974–76. Infected plants contained mostly cauliflower mosaic (CaMV) and turnip mosaic (TuMV) viruses but occasionally broccoli necrotic yellows virus (BNYV) was also found. The most seriously affected cultivar was the F₁ hybrid cv. Fasolt. Other hybrids including cvs Achilles, Citadel and King Arthur were more tolerant of infection. A key was developed for assessing the reactions of various F₁ hybrids and their parent inbred lines to CaMV + TuMV. Tests showed that the mean symptom severity of hybrids was usually less than the mean symptom severity of the parent inbreds, indicating that resistance to infection is partially dominant. The reactions of infected inbreds grown in winter under glasshouse conditions (15–18°C) were closely correlated with those of plants grown in the field. Thus, rapid selection of virus-tolerant inbreds can be made under glasshouse conditions.     

Selection for immunity in swede (Brassica napus) to infection by turnip mosaic virus

Fully heritable immunity to turnip mosaic virus (TuMV) was found in swede (Brassica napus) cvs Ruta Otofte and Bangholm. Attempts to ‘break’ this immunity were unsuccessful. The reactions of two immune lines of cv. Ruta Otofte to cauliflower mosaic virus, clubroot (Plasmodiophora brassicae) and powdery mildew (Erysiphe cruciferarum) were unchanged compared with the unselected parent line. A symptomless response to TuMV inoculation (possibly immunity) was also found in cv. Acme but not in the clubroot resistant cv. Marian. The percentage of plants which gave a symptomless response to TuMV inoculation differed greatly between cultivars and some seed stocks.     

Selective allele loss and interference between cauliflower mosaic virus DNAs

Summary Some cauliflower mosaic virus (CaMV) alleles are selectively lost during growth of the virus in mixedly infected turnip plants. Viral DNA from plants co-inoculated with DNA of the cabbage S isolate and infectious cabbage S DNA with an extra EcoRI restriciion site lacked the extra site. The EcoRI allele was also lost in most plants co-inoculated with a non-infectious mutant of cabbage S DNA while little selective allele loss was observed with two other non-infectious mutant DNAs. Plants co-inoculated with DNAs of closely-related isolates (CM4-184 and W) contained both parental viral DNAs and some DNAs with characteristics of both parents. Interference, scored as a reduced frequency of infection or a delay in symptom appearance relative to plants inoculated with wild-type DNA, occurred when plants were inoculated with wild-type and mutant DNAs covalently attached to one another in partial dimer plasmid DNAs. Similarities in the conditions leading to selective allele loss and those leading to interference suggest that both may have been due to active gene conversion between CaMV DNA molecules.     

The reactions of swede (Brassica napus) to infection by turnip mosaic virus

Turnip mosiac virus (TuMV) and cucumber mosaic virus (CMV) were the only viruses commonly isolated from naturally diseased swedes (Brassica napus) showing leaf mosaic and leaf and root necrosis. Only TuMV caused these symptoms when re-inoculated to swede. TuMV-infected plants showed a severe loss in leaf (55%) and root (63%) fresh weight after 140 days. Systemic leaf symptoms in infected swede plants varied greatly, but were predominantly necrotic (N) or mosaic (M). Plants were classified into one of seven reaction classes ranging from slight or severe necrosis, and mosaics with and without slight veinal necrosis. Swede cultivars differed markedly in their reaction to TuMV and contained different proportions of N- or M-reacting plants. The reactions of progeny of four resistant cv. Bangholm plants were separately inherited; progeny of two plants reacting either symptomlessly or necrotically and those of the other two plants developing mosaic symptoms only. Five isolates of TuMV from swede crops in different regions caused similar reactions but differed in virulence in the progeny of a self-pollinated resistant swede plant.     

Isolation of Viruses by Electro-Extraction of Infected Plants

The isolation of viruses from infected plant material by a process termed electro-extraction appeared to be a convenient and simple method of obtaining viruses in a fair state of purity. The method has the advantage over the conventional methods of virus purification that the infected plant tissue is not disintegrated and that organic solvents such as chloroform and butanol are avoided. The procedure used was demonstrated on the extraction of tobacco mosaic virus (TMV) from infected tobacco and turnip yellow mosaic virus (TYMV) from Chinese cabbage plants. To obtain the virus it was found advisable to freeze and thaw the plants prior to extraction.     

芜菁花叶病毒(TuMV)特性的研究进展

芜菁花叶病毒(TuMV)是世界范围内广泛分布的重要病毒。从生物学和理化特性、基因组结构、侵染和繁殖、变异及利用转基因技术提高病毒抗性等方面对TuMV的国内外研究现状进行了阐述。     

3. Isolation of Viruses by Electro-Extraction of Infected Plants

ABSTRACT

The isolation of viruses from infected plant material by a process termed electro-extraction appeared to be a convenient and simple method of obtaining viruses in a fair state of purity. The method has the advantage over the conventional methods of virus purification that the infected plant tissue is not disintegrated and that organic solvents such as chloroform and butanol are avoided. The procedure used was demonstrated on the extraction of tobacco mosaic virus (TMV) from infected tobacco and turnip yellow mosaic virus (TYMV) from Chinese cabbage plants. To obtain the virus it was found advisable to freeze and thaw the plants prior to extraction.