Tobacco Necrosis Research Articles

New Diseases and Epidemics Properties of Strains of Potato Virus YNin North America

A tobacco veinal necrosis strain of potato virus Y (PVY N ) was detected in tobacco production in Ontario in 1989. Subsequent surveys of potato production in Ontario, Prince Edward Island, New Brunswick, Nova Scotia, and Quebec and of table potatoes imported into Canada from California yielded similar isolates of PVY N . The host range and serological properties of several of these isolates were compared to a PVY N strain from Scotland and other PVY strains from the United States (PVY-NN, PVY-MN, and PVY-36) that had been reported to cause necrosis in tobacco (.)

Differential production of parasiticein, an elicitor of necrosis and resistance in tobacco, by isolates of Phytophthora parasitica

Fifteen isolates of Phytophthora parasitica, nine from tobacco (causing black shank disease) and six from other host plants, were compared by root inoculation with regard to their pathogenicity to young tobacco plants. A progressive invasion of the aerial parts over 1 week was observed only with the black shank isolates, while the non‐tobacco isolates induced leaf necrosis within 2 days. Similar necrosis occurred when the roots of tobacco plants were dipped in diluted culture filtrates from non‐tobacco isolates, but not in those from tobacco isolates. The necrosis‐inducing filtrates were shown to contain a c. 10‐kDa protein band which was not present in the other filtrates. This protein (named parasiticein) was purified by ion‐exchange chromatography to homogeneity in SDS‐PAGE and reverse‐phase HPLC. Parasiticein was serologically related to cryptogein, a member of the elicitin family of proteinaceous elicitors previously described from other Phytophthora species. Like the other elicitins, parasiticein induced necrosis in tobacco plants and protected them against black shank. It most closely resembled capsicein in being acidic and in inducing resistance at concentrations (10–100 μg per plant) that caused little leaf necrosis. It is suggested that the absence of parasiticein production by the black shank isolates might be a factor involved in their specific pathogenicity to tobacco.

Possible Nonpathogenic Origin of Hydrangea. Distortion

Florists’ hydrangea, Hydrangea macrophylla subsp. macrophylla var. macrophylla (Thunb.) Ser., is susceptible to a foliar malformation that appears during high day temperature (>30C) per iods of summer production. This condition previously has been termed hydrangea distortion (Weiler and Lopes, 1974). Affected plants produce thickened laminae that are sometimes mottled and reduced in width (Fig. 1). The plant apex becomes irregularly shortened, distorted apices often produce abnormal inflorescences, and many of the affected plants do not flower. In many respects, the symptoms of hydrangea distortion are similar to symptoms expressed by plants infected with a virus. Hydrangeas are known to be susceptible to nine viruses: alfalfa mosaic, cucumber mosaic, hydrangea ringspot, tobacco necrosis, tobacco rattle, tobacco ringspot, tomato ringspot, and tomato spotted wilt virus. A mycoplasma-like organism (MLO) also has been identified within hydrangeas (Kleinhempel et al., 1972; Lawson and Smith, 1980). The etiology of hydrangea distortion has not been described, and we therefore examined the possibility of a viral or MLO origin of this problem. Virus indicator plant species (Catharanthus roseus ‘Dwarf Bright Eyes’, Chenopodium amaranticolor, Chenopodium quinoa, Cucumis sativus, Gomphrena globosa ‘Buddy Purple’, Lycopersicon esculentum ‘Rutgers’, Nicotiana clevelandii, Nicotiana glutinosa, Nicotiana tabacum ‘Samsun NN’, Phaseolus vulgaris ‘Bountiful’, and Vigna sinensis ‘Early Ramshorn’) were inoculated with sap from distorted foliage of H. macrophylla ‘Rose Supreme’ or ‘Merritt’s Supreme’. Inoculum was prepared by grinding distorted leaves in

Protection Induced by Protein‐Lipopolysaccharide Complexes in Tobacco Leaves: Role of Protected Tissue Free‐space Solutes*

AbstractHypersensitive confluent necrosis in tobacco leaves, caused by Pseudomonas syringae pv. aptata, is prevented by an intercellular injection of protein‐lipopolysaccharide (pr‐LPS) complexes 48 h earlier. An increase (48 %) in the peroxidase activity and a new low molecular weight protein (ε 17.5 kD) were found in the intercellular fluid of protected tissue (treated fluid) 48 h after pr‐LPS infiltration. Up to 24 h after pr‐LPS infiltration the treated fluid did not inhibit the in vitro growth of heterologous bacteria. Injected into tobacco leaves 30 min before the bacteria, the treated fluid retarded the rate of intercellular bacterial growth between 8 and 12 h after infiltration of the bacteria compared with the control fluid. Up to 4 h after injection, the attachment of live and dead bacteria to the cell walls occurred in both the protected tissue and in the control; 4 h after injection, however, the dead bacteria were only weakly attached in the protected tissue. Within 20 min after the intercellular infiltration, there was a decrease in the heterologous bacterial number in the protected tissue, 33 % less than that in the control tissue.Protected tissue free‐space solutes did not have a direct antibacterial effect but activated an antibacterial response in the leaf tissue. Attachment of bacteria to the cell walls did not in itself trigger hypersensitive confluent necrosis. The protected tissue was initially more favourable for the survival of heterologous bacteria.

The Pathogenesis‐Related Proteins of Phsseolus Bean

AbstractSeven acidic low molecular weight pathogenesis‐related (PR) proteins from tobacco necrosis virus‐infected Phaseolus bean leaves were isolated, purified and partially characterized. The proteins could ba classified following the nomenclature of Antoniwet al. (1980) for tobacco PR proteins. Three groups are thus proposed, corresponding to molecular sizes of 16, 27 and 31 kD, very close to the values of three groups proposed for tobacco. Tentative correspondences between the PR proteins here described and those previously reported by other authors are also proposed.

Virus contamination of soils in forest ecosystems of the Federal Republic of Germany

AbstractInvestigations on virus contamination of soils in forests located in three federal states of West‐Germany have indicated that plant viruses are widely spread. From about one third of 284 samples mechanically transmissible viruses belonging to the potex‐, tobamo‐, tobacco necrosis‐ and potyvirus group were isolated by bait‐plant technique.

Chromatographic purification and characterization of elicitors of necrosis on tobacco produced by incompatible Phytophthora species

A chromatographic procedure was developed to purify the proteins eliciting necrosis in tobacco and produced in culture by three species of Phytophthora: cryptogein from P. cryptogea, cinnamomin from P. cinnamomi and capsicin from P. capsici. The procedure included ion-exchange chromatography and gel filtration on Sephadex G-25. Polyacrylamide gel electrophoresis data and the amino acid composition are in agreement with a molecular mass near 10 000 for these proteins. Their behaviour on ion-exchange columns indicates that cryptogein and cinnamomin are basic proteins and capsicin an acidic one. In biological tests on excised tobacco leaf, cryptogein and cinnamomin exhibited an activity 50–100-fold that of capsicin.

Changes in the Proteins of Bean Leaves Infected With Tobacco Necrosis or Alfalfa Mosaic Viruses

AbstractAcidic extracts from TNV and AMV infected “Saxa” bean leaves were electrophoretically examined for protein content. In native conditions of resolution (PAGE) at least three protein bands (PS1–3) not present in the control were found. In denaturing conditions (SDS–PAGE) at least one (PSa), but often two or three such proteins (PSb, c) were found in the same extracts.Chromatographic resolution of proteins on Sephadex G‐100 column resulted in partial purification of the PS‐proteins. Additional, not known before, slow‐migrating protein (PS0) induced by hypersensitive viral infection was discovered in some of the eluted fractions. Both PS0–3 and PSa–c proteins were present in the same fractions. This fact suggests their similarity in molecular weights and/or shapes.

Viruses and virus-like diseases affecting Betula spp

SynopsisSeven virus-like agents have been recognized naturally infecting birches in Europe or North America. Of these, only two have been studied in detail. Apple mosaic virus (AMV) infects Betula papyrifera and B. alleghaniensis in North America, and is associated with golden-white ring/line patterns in leaves. There is no evidence that AMV spreads between birches and no natural means of dissemination is known. In Europe, B. verrucosa and B. pubescens are naturally infected with cherry leaf roll virus (CLRV) which causes foliar mottle, pale yellow spots and bright green vein banding. In the United Kingdom, the prevalence of CLRV in unmanaged birch populations is less than in street trees. The virus is spread via birch seed and pollen which it infects and also contaminates. Sparse data suggest that the following viruses infect birch but their effects are unknown: yellow birch latent, tobacco rattle, tobacco necrosis, prunus necrotic ringspot and prune dwarf. There is a similar paucity of information concerning the cause of wood abnormalities that enhance the veneer value of affected timber: agents transmitted in soil or by grafting have been associated.

Characterisation of an Apparently Novel Soluble Protein Fraction found in Pathogen and Osmotically‐stressed Cucumber Cotyledons

AbstractCotyledons of cucumber (Cucumis sativus L. cv. Ashley) were used to study the alterations that occur in the soluble protein fraction following either tobacco necrosis virus‐elicited necrosis or a period of mannitol‐induced osmotic‐stress. One apparently novel fraction (termed γ) was induced in the soluble protein fraction after necrosis or osmotic stress and was identified by disc‐PAGE and gel staining. Smaller amounts of the γ fraction were present in apparently healthy tissue adjacent to necrotic halves of cotyledons (AH tissue), but were absent from any other leaves on these plants. The γ fraction is probably representative of at least three different proteins, two of which are constituents of control tissue (a glycoprotein and possibly one or two RNAse isozymes) while the third is a protein of unknown nature, present as the bulk of the γ fraction and possibly synthesized de novo.Following the radio‐active labelling of AH tissue it would appear that an increased protein synthesis, which is sensitive to the effects of translational inhibitors may result in the production of the γ fraction, but this is speculative. The major component of the γ fraction gave a single band on SDS PAGE, with an Mr of ca. 22,000 and focussed to a single band at pl 4,1 on IEF. While it is thought the bulk of the novel portion of the γ fraction is represented in these gel systems increased amounts of the constitutive glycoprotein component should be taken into consideration.Soluble extracts of protoplasts isolated from AH tissue retained only constitutional RNAse isozyme(s), and were totally devoid of the rest of the γ fraction. This result suggests that the other component(s) of the γ fraction is (are) subcellularly located in the plasmodesmata, between the cell wall and plasma membrane or non‐covalently attached to either.

Lack of specificity of virus-stimulated plant RNA-dependent RNA polymerases

RNA-Dependent RNA polymerase preparations from tabacco and cowpea plants, either uninfected, or infected with tobacco necrosis or cowpea mosaic virus, were tested for their capability to bind radioactive viral or other RNAs or polynucleotides. The limited binding of up to 30% of all RNAs tested was found to be completely nonspecific in regard to any infecting virus, and it was nonspecifically diminished by excess of other unlabeled RNAs. These data are in line with our previous conclusion that plant viral RNAs are replicated by host enzymes. The data also speak against the hypothesis that these enzymes might be modified as a consequence of viral infection.

Prevention of confluent hypersensitive necrosis in tobacco leaves by a bacterial protein-lipopolysaccharide complex

A protein-lipopolysaccharide (pr-LPS) complex has been selectively detached from washed living cells of Erwinia chrysanthemi NCPPB 454 by treatment with disodium ethylene diamine tetraacetate (EDTA). The material was partially purified by ultrafiltration, then chemically characterized, and its purity was checked by spectrophotometry and gel chromatography. The method yielded a protein-lipopolysaccharide complex of the outer membrane containing about 15% protein. The protein moiety was characterized by a major polypeptide of 40 000 daltons. Dispersions of the complex were injected into interveinal areas of mature tobacco leaves kept at 25°C. A slight chlorosis slowly developed in most treated areas. After 48 h, treated and control areas were injected with 4 × 10 7 cells/ml Pseudomonas syringae NCPPB 2664. Whereas the control areas developed confluent hypersensitive necrosis, the areas pretreated with the complex did not. Prevention of hypersensitivity was consistent at concentrations of 50 to 500 μg/ml; complete or partial prevention was obtained in the range of 3 to 50 μg/ml. Substances immunologically identical to the purified pr-LPS complex were spontaneously released by E. chrysanthemi during growth in broth cultures. The results thus suggest that pr-LPS complexes of bacterial outer membranes may play a rôle in modifying the response of plants to phytopathogenic bacteria.

Physiochemical and serological properties of a virus from saguaro cactus

The molecular weight of a spherical virus from saguaro cactus (SV) was estimated to be 7.9 × 10 6 from a sedimentation coefficient of 118 S and a diffusion coefficient of 1.22 × 10 −7 cm 2/sec. SV was found to contain 17.2% RNA, based on its phosphorus content of 1.66% and the nucleotide composition of its nucleic acid (29% guanylic acid, 24% adenylic acid, 21% cytidylic acid, and 27% uridylic acid). A single RNA component, a major protein component (90–95%), and two minor protein components were detected by electrophoresis in polyacrylamide gels and their molecular weights were estimated at 1.4 × 10 6, 38,900, 90,200, and 29,400, respectively. At pH 4, 5, 6, and 7, SV had three electrophoretic components, at pH 8 only two. The three components at pH 7 had similar amino acid compositions. SV was dissociated by low levels of sodium dodecyl- sulphate (0.05% at pH 7 and 0.10% at pH 5) and is probably held together by protein-RNA interactions. SV was not related serologically to sowbane mosaic, southern bean mosaic, tobacco necrosis, carnation mottle, turnip crinkle, tomato bushy stunt, cucumber necrosis, or carnation ringspot viruses.

Incorporation of Uridine‐H3 into Healthy and Tobacco Necrosis Virus‐infected Mesophyll Cells of Chenopodium amaranticolor1)

Incorporation of Uridine‐H³ into Healthy and Tobacco Necrosis Virus‐infected Mesophyll Cells of <i>Chenopodium amaranticolor</i>¹)

Hypersensitive resistance. The role of cell wall glycoproteins in virus localization

Glycoprotein complexes were extracted from Phaseolus vulgaris var. Pinto infected with tobacco mosaic virus (TMV) or with tobacco necrosis, or after sham inoculation. After all three treatments there were two distinct periods when glycoproteins were at a maximum. The product accumulated during the primary response contained both β(1 → 3)- and β(1 → 4)-linked glucopyranosyl residues. That of the secondary response contained mainly β(1 → 4)- but no β(1 → 3)-linked residues. These two glycoprotein complexes were absent from healthy non-inoculated plants.The similarity between results from sham-inoculated plants and those inoculated with virus suggests that the glycoprotein formation is a wound response, induced through mechanical inoculations and not through virus infection. Deposition of glycoproteins within the cell walls in advance of infection may localize the virus by reducing the number of intercellular pathways for virus spread. If virus has already spread beyond the zone of cell wall modifications, localization will be incomplete.

Peroxidase isoenzymes in leaves of cucumber ( Cucumis sativus L.) cultivars systemically infected with the W strain of cucumber mosaic virus

The isoperoxidases in leaf extracts of healthy and cucumber mosaic virus infected cucumber cultivars of differing susceptibility to infection and of healthy tobacco plants have been examined by disk electrophoresis. The relative intensities of the principal isoperoxidases in zymograms of extracts from systemitically infected cucumber leaves, compared with those of healthy control leaves, were correlated with symptom severity and peroxidase activity. Thus, extracts from infected leaves of the susceptible cultivars Ashley and Marketer, with pronounced symptoms, showed greater intensities of the principal isoperoxidase bands in comparison with extracts of the resistant, almost symptomless cultivar China, and had greater peroxidase activities. The changes in isoperoxidase patternsappeared to be nonspecific responses to infection, although zymograms of extracts from senescing detached leaves of the four cucumber cultivars were not identical to those from virus infected plants. Cucumber mosaic virus infection of both cucumber and tobacco cultivars resulted in increased activity or stimulated synthesis of host isoperoxidases rather than induction of virus specific ones, in support of conclusions reached by other workers, based on experiments with several viruses including tobacco mosaic, tobacco streak, tobacco necrosis and tobacco ringspot viruses.

Ammonia as a necrotoxin in the hypersensitive reaction caused by bacteria in tobacco leaves

Tobacco leaves inoculated with 108 cells/ml of the incompatible bacteria Pseudomonas pisi, P. syringae, P. lachrymans, or Erwinia amylovora developed hypersensitive tissue necrosis, on the first day after inoculation. Similar tissue necrosis developed on the leaves inoculated with the compatible bacterium P. tabaci on the second day. By the time the symptoms developed ammonia gas had evolved from the inoculated leaves. Tissue necrosis caused by bacteria could be reproduced by exposing healthy tobacco leaves to ammonia gas. The amount of ammonia evolved during the development of either the hypersensitive reactions or the disease was enough to account for the formation of tissue necrosis in tobacco.

Tissue Necrosis in Tobacco Caused by a Saprophylic Bacterium

Tissue Necrosis in Tobacco Caused by a Saprophylic Bacterium

Isolation of a virus inhibitor from plants with localized infections

Systemic resistance was induced in the leaves of Datura stramonium L., Nicotiana glutinosa, Phaseolus vulgaris var. Prince, and Chenopodium amaranticolor by localized infection with tobacco mosaic or necrosis viruses (TMV, TNV). Crude extracts prepared from the resistant leaves were more inhibitory to TNV infection on Phaseolus than were extracts from non-resistant (control) leaves. Subsequently, crude extracts were fractionated into protein and ribonucleic acid (RNA) and inhibitory activity from resistant areas was retained in the RNA preparation. The RNA from resistant and non-resistant leaves was purified and separated into three fractions (about 27 S, 17 S, and 3 S), by gel filtration on Sephadex G-200. The 27-S and 17-S fractions from both treatments inhibited TNV infection by similar amounts. However, the 3-S RNA from resistant leaves reduced lesion number to a greater extent when compared to both the 3-S RNA from control leaves and the 27-S and 17-S RNA from either treatment. The lowest molecular weight RNA isolated from systemic-induced resistant areas gave positive tests with orcinol reagent and had an absorption spectrum characteristic of a nucleic acid. When treated with pancreatic ribonuclease and mixed with TNV, inhibition no longer occurred.

The use of negative staining in the electron microscopic examination of plant viruses in crude extracts

Extracts from the leaves of plants infected with a wide range of viruses were stained with potassium phosphotungstate and examined in the electron microscope in an attempt to detect virus particles. Most specimens were made by applying a strip of tissue, peeled from the underside of a leaf, to a drop of stain on an electron microscope grid. The rod-shaped particles of barley stripe mosaic virus, tobacco rattle virus, tobacco mosaic virus, potato virus X, potato virus M, and potato virus Y could be detected easily. Measurement showed that, as in the shadow-cast preparations of other workers, the lengths of the great majority of particles of each virus fell within one, or with tobacco rattle virus within two, narrow ranges. For each virus the mean length of the particles within such a range was usually closely similar to the value obtained by others using shadowed preparations. Of the viruses with isometric particles, turnip yellow mosaic, wild cucumber mosaic, turnip crinkle, and broad bean mottle virus particles were usually easy to detect, principally because of the large numbers present. In preparations of turnip yellow mosaic virus round particles, larger than those of the virus and usually “empty” were occasionally present. Somewhat similar large “empty” particles were common in preparations of wild cucumber mosaic virus and frequently such a large particle enclosed an “empty” virus particle. Careful examination was necessary to detect particles of the viruses of cucumber mosaic, lucerne mosaic, tobacco necrosis, and tobacco ringspot, either because few particles were present or because they were poorly contrasted against the background.