Effect of thermal shock treatments on symptom expression in test plants inoculated with potato aucuba mosaic virus

Potato aucuba mosaic virus (PAMV) has few reliable local lesion assay hosts. However, lesions formed when PAMV-inoculated leaves were exposed to thermal shock (dipping for 40 s in water at 50 or 2 °C). Leaves of Nicotiana tabacum (cv. Xanthi-nc, Samsun or Samsun NN), Hyoscyamus niger and Datura metel consistently developed necrotic lesions, leaves of Chenopodium amaranticolor developed whitish rings, and leaves of N. glutinosa developed diffuse cream-coloured rings and spots. In PAMV-inoculated leaves of Xanthi-nc tobacco, C. amaranticolor and D. metel, lesions formed only in areas exposed to light. Thermal shocks applied to systemically infected leaves of Xanthi-nc tobacco induced necrotic vein banding patterns. In inoculated Xanthi-nc tobacco leaves, PAMV seemed confined to local lesions. The rate of lesion enlargement was therefore a measure of rate of virus spread. Lesion size increased as the interval between inoculation and shock treatment increased. The mean rates of increase in lesion radius were 17 and 27 Cμm/h at 15 and 25°C respectively. ‘Target’ lesions, composed of concentric necrotic rings, formed when inoculated Xanthi-nc tobacco leaves were given two or more 50°C shocks. The first of two 50°C treatments decreased subsequent rates of lesion enlargement.     

Inhibitory effect of fucoidan from brown alga Fucus evanescens on the spread of infection induced by tobacco mosaic virus in tobacco leaves of two cultivars

The effect of fucoidan from the brown alga Fucus evanescens on the spread of infection induced by tobacco mosaic virus (TMV) was investigated in the leaves of tobacco (Nicotiana tabacum L.) of two cultivars (Ksanti-nk and Samsun). In the leaves of cv. Ksanti-nk inoculated with a mixture of TMV preparation (2 μg/ml) and fucoidan (1 mg/ml), the number of local necrotic lesions induced by the virus decreased by more than 90% as compared with the leaves inoculated with the virus alone. In tobacco leaves of cv. Samsun, virulence and the concentration of the virus 3 days after inoculation with the same mixture of TMV and fucoidan were by 62 and 66%, respectively, lower than in the leaves inoculated with TMV alone. As the infection spread, the inhibitory effect of fucoidan decreased. When the leaves were treated with fucoidan before and after the inoculation with TMV, its antiviral activity was less pronounced than when a mixture of the virus and the polysaccharide was used as inoculum. Electron microscopic investigation of TMV mixed with fucoidan often showed agglutinated virions. The highest virulence of the mixture (TMV preparation, 12 μg/ml, plus fucoidan, 1 mg/ml) was observed upon its twofold dilution, and after that it decreased. It was concluded that, when the leaves were inoculated with the mixture of TMV and fucoidan, the latter affected not only the plant but the virus as well. Treatment of tobacco leaves, cv. Ksanti-nk, with actinomycin D (10 μg/ml) 24 h before the inoculation with TMV almost completely suppressed the effect of fucoidan, indicating that fucoidan acted at a gene level.     

Can Salicylic Acid Affect the Intercellular Transport of the Tobacco Mosaic Virus by Changing Plasmodesmal Permeability?

We studied the effects of salicylic acid (SA) on the plasmodesmal permeability as evaluated by the tobacco mosaic virus (TMV) spreading in tobacco Nicotiana glutinosaleaves, where TMV induces necrotic lesions. When leaves were treated with SA simultaneously with their viral inoculation, SA retarded the development of necrotic lesions and reduced their number. When inoculated leaves were kept on the SA solution at an elevated temperature (31°C) for a short period of time, the size of the necrotic lesions, which developed after leaf transfer to room temperature, was decreased. SA stimulated the formation of rapid callose involved in the control of the plasmodesmal permeability, which was assessed from fluorescence after tissue staining with Aniline Blue. On the basis of these data, we suggest that SA suppressed TMV spreading in the inoculated tobacco leaves by reducing the plasmodesmal permeability.     

The Association of "Pathogenesis-related" Proteins with Viral Induced Necrosis in Nicotiana sylvestris

The association of “pathogenesis-related” (PR) proteins with protection from superinfection, systemic acquired resistance and production of localized necrotic lesions was examined with a system using tobacco mosaic virus (TMV) and Nicotiana sylvestris. Leaves of N. sylvestris with a mosaic from earlier inoculation with a systemically infecting strain of TMV (TMV-C) and control plants were challenged with a necrotizing strain of TMV (TMV-P), RNA of TMV-P and turnip mosaic virus (TuMV). TMV-P virions produced localized necrotic lesions only in the dark green areas of the mosaic of TMV-C infected plants. Both RNA of TMV-P and TuMV produced localized necrotic lesions in both light green and dark green areas of the mosaic of TMV-C infected plants. All three challenge inocula produced localized necrotic lesions in previously uninoculated plants. Six days after challenge inoculation proteins were extracted from separated dark green and light green mosaic leaf tissue, and leaf material from control plants. Proteins were separated by electrophoresis in a 5 % polyacrylamide spacer gel and 10 % polyacrylamide running gel. PR proteins were found in tissue where localized necrotic lesions were produced as a result of challenge inoculation, but not in tissue that was not superinfected. PR proteins were not found in light green or dark green mosaic leaf tissue as a result of TMV-C inoculation. No PR proteins were evident in protein extracts from light green tissue challenged with TMV-P, although PR proteins were produced in dark green tissue, where necrosis occurred, from the same leaves. Systemic acquired resistance (reduction in size of lesions formed by a challenge inoculation) to TuMV or RNA of TMV-P and PR protein concentration was measured at various times in light green areas of mosaic leaves where dark green areas of the mosaic leaves had been inoculated with TMV-P. No quantitative or temporal relationship between the onset of resistance and PR protein production was found. It is concluded that PR proteins are a result of pathogen induced necrosis and not significantly involved in the mechanism(s) of viral induced resistance.     

Host range and some properties of potato mop-top virus

Potato mop-top virus (PMTV) was transmitted by inoculation of sap to twenty-six species in the Solanaceae or Chenopodiaceae and to Tetragonia expansa; species in eleven other plant families were not infected. The virus was cultured in inoculated leaves of Nicotiana tabacum cv. Xanthi-nc or in N. debneyi. Diagnostic local lesions were produced in Chenopodium amaranticolor. In winter, ten solanaceous species were slowly invaded systemically but the first leaves infected were those immediately above inoculated leaves. When transmitted to Arran Pilot potato by the vector Spongospora subterranea, PMTV induced all the main types of shoot and tuber symptoms found in naturally infected plants. Isolates of PMTV from different sources differed considerably in virulence. PMTV-containing tobacco sap lost infectivity when heated for 10 min at 80 °C, diluted to 10^-4, or stored at 20 °C for 14 weeks. Infectivity was partially stabilized by 0·02% sodium azide. When sap was centrifuged for 10 min at 8000 g, infectivity was mainly in the sediment. Infective sap contained straight rod-shaped particles about 20 nm wide, with lengths up to 900 nm and crossbands at intervals of 2·5 nm. Many of the particles were aggregated side-to-side, and the ends of most seemed damaged. The slight infectivity of phenol-treated leaf extracts was abolished by pancreatic ribonuclease. The present cryptogram of PMTV is R/*:*/*:E/E:S/Fu.     

The Effects of Aspirin and Polyacrylic Acid on the Multiplication and Spread of TMV in Different Cultivars of Tobacco with and without the N-gene

Aspirin treatment of leaves of Nicotiana tabacum cv. Samsun at 20°C induced PR-proteins and reduced the amount of tobacco mosaic virus (TMV) accumulated 7 days after inoculation. However, at 32°C both the amount of PR-proteins induced and the reduction of TMV accumulated were less. Polyacrylic acid did not induce PR-proteins, and caused little or no reduction in the amount of TMV accumulated at 20°C. In cv. Samsun NN at 32°C. aspirin induced the PR-proteins and reduced the spread of TMV to surrounding tissue as treasured by the size of lesions produced on subsequent transfer to 20°C. Polyacrylic acid did not induce PR-proteins in Samsun NN and had no effect on the spread of TMV. In cv. Xanthi-ne, at 32°C aspirin and polyacrylic acid induced PR-proteins and reduced the spread of TMV. At 35°C, polyacrylic acid induced little or no PR-proteins and did not affect the spread of TMV.     

Influence of κ/β-carrageenan from red alga <Emphasis Type="Italic">Tichocarpus crinitus</Emphasis> on development of local infection induced by tobacco mosaic virus in Xanthi-nc tobacco leaves

The influence of κ/β-carrageenan from red marine alga Tichocarpus crinitus on the development of tobacco mosaic virus (TMV) infection in Xanthi-nc tobacco leaves was studied. It was shown that the number of necrotic lesions on the leaves inoculated with the mixture of TMV (2 μg/ml) and carrageenan (1 mg/ml) was reduced by 87%, compared to the leaves inoculated with the virus only. The suppression of virus infection was also observed when leaves were treated with carrageenan 24 h before or 24 h after leaf inoculation with TMV; however, in these cases, suppression was less evident than after inoculation with the virus-polysaccharide mixture. It is supposed that the antiviral activity of carrageenan applied together with TMV may be explained by its action not only on the plant but also on the virus itself. The inhibitory effect of carrageenan pretreatment can be explained by its favorable effect on tissue resistance to infection. The suppression of this resistance by actinomycin D indicates that carrageenan functions via its action on the cell genome.     

Studies on Induced Resistance to Tobacco Mosaic Virus, in Samsun NN Tobacco and Changes in Ribosomal Fractions

Resistance to tobacco mosaic virus (TMV) was activated by various forms of induction in Samsun NN tobacco leaves, and the intensity of the different forms was compared. Induced resistance was highest in leaf tissue between TMV inoculated stripes parallel to the mid-vein and after injection of ethylene maleic anhydride copolymer (EMA), followed by that induced in distal half leaves after inoculating the basal halves with TMV. Resistance in upper leaves following inoculation of the lower leaves with TMV was relatively low, while induction due to lesions caused by ethrel gave an intermediate degree of resistance. Estimation of resistance by size and number of local lesions was correlated with the amount of extractable virus as measured by enzyme-linked immunosorbent assay (ELISA), thus indicating that in the resistant tissue virus replication, and not only the development of necrotic local lesions, is suppressed. An increase in a specific ribosomal fraction (R₂), recovered by a two-step procedure, was observed in tissues where resistance was most intense, i.e., between TMV stripes and after EMA injection. It may be that this specific ribosomal fraction participates in maintaining the resistant state.     

Constitutively Elevated Levels of Putrescine and Putrescine-Generating Enzymes Correlated with Oxidant Stress Resistance in Conyza bonariensis and Wheat

Changes in ascorbate and glutathione levels and in activities of ascorbate peroxidase, catalase, dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione S-transferase (GST), and superoxide dismutase (SOD) were investigated in tobacco mosaic virus (TMV)-inoculated lower leaves and in non-inoculated upper leaves of Nicotiana tabacum L. cv Xanthi-nc. In separate experiments the effects of exogenous salicylic acid (SA) were also studied. Symptom appearance after TMV inoculation was preceded by a slight, transient decline of ascorbate peroxidase, GR, GST, and SOD activities in the inoculated lower leaves, but after the onset of necrosis these activities and the glutathione level substantially increased. Ascorbic acid level and DHAR activity declined and dehydroascorbate accumulated in the inoculated leaves. In upper leaves, the glutathione level and the activities of GR, GST, and SOD increased 10 to 14 d after TMV inoculation of the lower leaves, concomitantly with the development of systemic acquired resistance. From the six distinct SOD isoenzymes found in tobacco leaves, only the activities of Cu,Zn-SOD isoenzymes were affected by TMV. SA injection induced DHAR, GR, GST, and SOD activities. Catalase activities were not modified by TMV infection or SA treatment. It is supposed that stimulated antioxidative processes contribute to the suppression of necrotic symptom development in leaves with systemic acquired resistance.     

Demonstration of the Specific Involvement of Coat Protein in Tobacco Mosaic Virus (TMV) Cross Protection using a TMV Coat Protein Mutant

The DT-1G mutant of tobacco mosaic virus (TMV) which has no coat protein was used to study the specific involvement of coat protein in TMV cross protection in N. sylvestris. Leaves of N. sylvestris previously inoculated with the mutantor the common strain of TMV were challenged with either turnip mosaic virus (TuMV) or a strain of TMV (TMV-N). Both TuMV and TMV-N produce necrotic lesions on N. sylvestris. About one-half as many lesions were produced by TuMV and TMV-N on leaves, inoculated with the DT-1G mutant compared with lesions produced by the same inoculum on control leaves. When leaves of N. sylvestris previously inoculated with the common strain of TMV were challenged with either TuMV or TMV-N, TuMV produced about one-half as many lesions as on control leaves whereas TMV-N produced about one-tenth as many lesions as on control leaves. A high level of non-specific resistance was induced by the mutant without coat protein, but it did not specifically protect against TMV.     

Induction of Systemic Resistance to Tobacco Powdery Mildew by Tobacco Mosaic Virus, Tobacco Necrosis Virus or Ethephon

Local infections of either TMV or TNV in tobacco plants cv. Havana 425 (hypersensitive to TMV) proved effective in inducing systemic resistance to subsequent inoculation with the powdery mildew fungus Erysiphe cichoracearum DC. The proportion of leaf surface invaded by this pathogen and the amount of conidia it produced were both significantly lower in virus inoculated plants than in non-inoculated controls. However, the decrease in sporulation rate was less regularly observed than the reduction in leaf area infected. TMV was more effective than TNV in protecting tobacco plants from powdery mildew. E. cichoracearum is thus added to the list of challenge pathogens to which TMV or TNV are known to induce resistance in the host plants. Necrotic lesions caused to the leaves by local treatment with Ethephon (an ethylene-releasing compound) also conferred to tobacco some degree of systemic resistance to the same fungal pathogen, more frequently visible as a reduction of leaf area invaded. The protection due to the Ethephon lesions was in present experiments less marked than that of TMV. No effects against subsequent powdery mildew infection were obtained when point freeze necrotic lesions were provoked on the plants.     

Ornithine decarboxylase activity and the hypersensitive reaction to tobacco mosaic virus in Nicotiana tabacum

The activity of ornithine decarboxylase (ODC) is increased 20 fold in leaves of Nicotiana tabacum cv. Xanthi n.c. following infection with tobacco mosaic virus at 20°. The activity reaches its maximum when localized necrotic lesions appear. There is little or no increase in plants kept at 32° when infection is systemic. However, if the infected plants are transferred to 20°, a marked and rapid increase in ODC activity occurs in the upper leaves, which collapse seven to nine hours after the transfer. ODC activity therefore parallels the activity of phenylalanine ammonia lyase during the hypersensitive reaction. Tyrosine decarboxylase was found to be activated in the same conditions. By contrast no increase in arginine decarboxylase activity could be detected. Temperature has a much greater effect on the polyamine and tyramine content of Xanthi n.c. leaves than does infection with TMV.     

Effect of Temperature on Susceptibility of the Primary Leaves ofPhaseolus vulgaris L. to Red Clover Mottle Virus

Red clover mottle virus isolated in Czechoslovakia was studied in relation to its reaction to varying temperature on primary French bean leaves (Phaseolus vulgaris L.) on which it forms local necrotic lesions. The plants were kept 24 or 48 h before, or 24 or 48 h after inoculation at the temperatures 23, 25, 27, 30, 33 and 36°C. After such exposures the French beans were kept at a constant temperature of 25°C. The lesions were counted at various intervals. In the experiment the optimal temperature for the maximum number of lesions seems to be 36°C 48 h before inoculation. The temperature above 25°C applied 24 h after inoculation seems to have a decreasing effect upon the number of lesions formed by RCMV on primary leaves of French beans and the lesions appeared several hours later, especially at 30, 33 and 36°C. The temperatures 27, 30 and 33°C applied 48 h after inoculation have a further decreasing effect on the number of lesions. The temperature of 36°C applied 48 h after inoculation has an inactivating effect upon RCMV inoculated on French bean leaves and no lesions appeared 5 days after inoculation.     

Xanthine Oxidase Activity in the Susceptible and Hypersensitive Responses of Tobacco Leaves to Tobacco Mosaic Virus Infection

A superoxide-producing xanthine oxidoreductase was isolated and quantified after polyacrylamide disc gel electrophoresis of tobacco leaf extracts. The results obtained indicate that, like uricase activity, a slight increase in tobacco xanthine oxidase activity takes place in the susceptible interaction with tobacco mosaic virus (TMV). In contrast, out of three hypersensitive tobacco cultivars tested, only two showed the same slight increase m activity during the late stage of hypersensitive response.
Allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidine] a specific and potent in vitro and in vivo inhibitor of xanthine oxidoreductase, applied to tobacco plants by root absorption, starting about 8 days before the inoculation, did not affect the hypersensitive response but weakened the hypersensitivity-linked virus localization and promoted the movement of a certain amount of TMV particles and/or virus related material from necrotic lesions which induced systemic necrotic symptoms in uninoculated leaves. However, due to the inefficacy of allopurinol in preventing necrotic lesion development, all results are consistent with the hypothesis that xanthine oxidoreductase, the first enzyme in purine oxidative degradation, plays only a secondary role during induction of primary hypersensitive cell death in TMV infected tobacco leaves.     

The Effect of Post Inoculation Treatment with Mineral Oil on TMV Multiplication in Tobacco Leaves and Protoplasts

Half leaves of N. tabacum dipped into a 0.2 % emulsion of meneral oil 15min after inoculation with tabacco masaic virus (TMV) developed significantly fewer lesion than when not dipped Dipping reduced lesion numbers when applied up to 2½ h after inoculation and multiple dippings were more effective than one. The effect of oil was the same whether the inoculum was whole virus or RNA. Tobacco protoplasts treated with mineral oil contained less virus than untreated protoplasts. The oil probably acted by killing the protoplasts and was effective only when protoplasts were centrifuged through the oil emulsion, When water-treated leaves were dipped into a TMV solution there was an effect on TMV infection similar to that caused by dipping in oil after TMV inoculation.     

Induction of Benzoic Acid 2-Hydroxylase in Virus-Inoculated Tobacco

Salicylic acid (SA) plays an important role in the induction of plant resistance to pathogens. An accompanying article (N. Yalpani, J. Leon, M.A. Lawton, I. Raskin [1993] Plant Physiol 103: 315-321) shows that SA is synthesized via the decarboxylation of cinnamic acid to benzoic acid (BA), which is, in turn, hydroxylated to SA. Leaf extracts of tobacco (Nicotiana tabacum L. cv Xanthi-nc) catalyze the 2-hydroxylation of BA to SA. The monooxygenase catalyzing this reaction, benzoic acid 2-hydroxylase (BA2H), required NAD(P)H or reduced methyl viologen as an electron donor. BA2H activity was detected in healthy tobacco leaf extracts (1-2 nmol h-1 g-1 fresh weight) and was significantly increased upon inoculation with tobacco mosaic virus (TMV). This increase paralleled the levels of free SA in the leaves. Induction of BA2H activity was restricted to tissue expressing a hypersensitive response at 24[deg]C. TMV induction of BA2H activity and SA accumulation were inhibited when inoculated tobacco plants were incubated at 32[deg]C. However, when inoculated plants were incubated for 4 d at 32[deg]C and then transferred to 24[deg]C, they showed a 15-fold increase in BA2H activity and a 65-fold increase in free SA content compared with healthy plants incubated at 24[deg]C. Treatment of leaf tissue with the protein synthesis inhibitor cycloheximide blocked the induction of BA2H activity by TMV. The effect of TMV inoculation on BA2H could be duplicated by infiltrating leaf discs of healthy plants with BA. This response was observed even when applied levels of BA were much lower than the levels observed in vivo after virus inoculation. Feeding tobacco leaves with phenylalanine, cinnamic acid, or o-coumaric acid (putative precursors of SA) failed to trigger the induction of BA2H activity. BA2H appears to be a pathogen-inducible protein with an important regulatory role in SA accumulation during the development of induced resistance to TMV in tobacco.     

STUDIES ON THE EFFECT OF TEMPERATURE ON VIRUS MULTIPLICATION IN INOCULATED LEAVES

The rate at which the Rothamsted tobacco necrosis virus (RTNV) accumulates in inoculated French bean leaves increases with rising temperature to 22°C. and then decreases. Three days after inoculation, leaves at 22°C. contain 4000 times as much virus as at 10°C. and 1000 times as much as at 30°C. At all temperatures the rate of accumulation may depend on the balance between synthesis and inactivation of RTNV, but inactivation becomes increasingly important with rise of temperature above 22° C. and as the virus content of the leaves increases. Above 22°C. the rate of multiplication may increase but less rapidly than the rate of inactivation, and exposing inoculated leaves to ultra-violet radiation at various intervals after inoculation suggests that at 30°C. RTNV multiplies in and moves from the initially infected epidermal cells in slightly less than the 6 hr. needed at 22°C. Thirty hr. are needed at 10°C. Newly formed virus is rapidly inactivated at 30°C. Raising the ambient temperature also decreases the numbers of local lesions produced by RTNV, possibly by increasing the chances that the introduced virus particles will become inactivated. Increasing the virus content of the inoculum above the level giving one lesion per sq.cm. does not increase the subsequent virus content of inoculated leaves.
At temperatures of 30°C. and below, tomato aucuba mosaic virus produces necrotic lesions in leaves of tobacco and Nicotiana glutinosa whereas above 30°C. the lesions are chlorotic. In both hosts this virus multiplies more rapidly when the infected cells are killed.     

Effects of light and temperature on symptom development and virus content of tobacco leaves inoculated with potato mop-top virus

Necrotic spots or small rings develop after 3–4 days in leaves of Nicotiana tabacum cv. Xanthi-nc inoculated with potato mop-top virus and kept at 14 °C in continuous light (4320 lux); a series of concentric necrotic rings of increasing diameter then form at 2- to 3-day intervals around each initial lesion. Successive rings take longer to appear when either the light intensity or the photoperiod is decreased. Virus accumulation is much decreased and lesions rarely develop either at 14· in darkness or at 22° in light. Virus accumulates rapidly when plants are transferred from these conditions to 14° in light (4320 lux), and necrotic spots or rings develop whose size depends on the interval between inoculation and transfer, and on the conditions during this period. In such plants, necrosis seems to occur only when conditions become favourable for virus synthesis, it is confined to recently infected cells and it does not prevent virus spread to further healthy cells. From the sizes of the necrotic rings, the virus is estimated to invade tissue in light (4320 lux) at c. 38 μm/h at 22° and c. 16 μm/h at 14°. Invasion in darkness at either temperature is very slow. Necrotic rings develop, and the rate of virus accumulation increases when inoculated plants are transferred from 22° in light (4320 lux) to 14° in darkness, but no lesions appear when the order of the treatments is reversed. The process of lesion formation thus includes an early phase requiring light and a later phase requiring low temperature. The light-requiring phase takes about a day at 14° but less at 22°. The later phase takes about 2 days in light (4320 lux) or 3 days in darkness.