V. P. Nagorskaya

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.

Effect of κ/ß-Carrageenan from red alga Tichocarpus crinitus (Tichocarpaceae) on infection of detached tobacco leaves with tobacco mosaic virus

It is shown that κ/ß-carrageenan obtained from red marine alga Tichocarpus crinitus possesses antiviral activity against Tobacco mosaic virus (TMV). Two days after inoculation of Nicotiana tabacum L. cv. ‘Samsun’ detached leaves with a mixture of TMV (1 μ/ml) and carrageenan (1 mg/ml), the infectivity and virus content in the leaves as well as the intracellular accumulation of virus particles were significantly inhibited in comparison with leaves inoculated with TMV only. The carrageenan applied 24 h prior to inoculation with TMV also exhibited the antiviral effects but to a lower degree than when the virus and polysaccharide were inoculated together. As the infection developed, the inhibitory effects of carrageenan decreased. Treatment of the leaves with carrageenan affected the formation of TMV-specific intracellular inclusions (granular and tubule) known to consist of the virus-encoded protein components of the viral replicase. A characteristic of the infection development in the presence of carrageenan, 4 days after infection of the leaves with TMV, was the predominant formation in cells of granular inclusions famous to appear at early stages of TMV multiplication. At the same time, cells of the leaves inoculated with TMV alone contained for the most part tubule inclusions considered to arise from granular ones on the advanced infection stages. Taken together, our observations indicate that the κ/ß-carrageenan inhibits TMV infection in detached tobacco leaves at early stages.ZusammenfassungEs wurde gezeigt, dass κ/ß-Karagheen, das aus den roten Meeresalgen Tichocarpus crinitus gewonnen wurde, hohe Antivirusaktivität gegen das Tabakmosaikvirus (TMV) besitzt. Zwei Tage nach der Inokulation der Blätter von Nicotiana tabacum L. cv. ‘Samsun’ mit der Mischung TMV (1 μ/ml) und κ/ß-Karagheen (1 mg/ml) werden die Infektionsstufe, der Gehalt an Virus in den Blättern, und auch die Akkumulation von Virusbestandteilen in den Zellen signifikant gehemmt. κ/ß-Karagheen, das 24 h vor der Inokulation mit TMV angewendet wurde, auch Antiviruseffekte, aber weniger als wenn Virus und Polysaccharid gemeinsam inokuliert wurden. Im Laufe der Entwicklung der Infektion wurden die Hemmeffekte von κ/ß-Karagheen immer schwächer. Die Behandlung der Blätter mit κ/ß-Karagheen beeinflusste die Entstehung von TMV-spezifischen intrazellulären Komponenten (granulär und tubulär), die bekanntlich aus den virus-codierten Eiweißkomponenten der Virenreplikase bestehen. Charakteristisches Merkmal der Entwicklung der Infektion in Anwesenheit von κ/ß-Karagheen (4 Tage nach der Inokulation der Blätter mit TMV) war die vorwiegende Bildung der Granular- komponenten in den Zellen, die in den Frühstadien der TMV-Replikation entstehen. Die Zellen der Blätter, die nur mit TMV inokuliert worden waren, enthielten hauptsächlich die tubuläre Komponente, die, wie man vermutet, sich während der fortschreitenden Etappen der Infektion aus den Granular- komponenten bilden. Die Ergebnisse zeigen, dass κ/ß-Karagheen die TMV-Infektion in abgeschnittenen Tabakblättern hemmt.

Influence of κ/β-carrageenan from red alga Tichocarpus crinitus 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.

Enzymatic transformation of biologically active 1,3;1,6-β-D-glucan. Structure and activity of resulting fragments

The fragmentation of the biologically active 1,3;1,6-β-D-glucan Antivir by endo-1,3-β-D-glucanase LIV from crystalline styles of the marine mollusk Spisula sachalinensis was carried out. It was found that low molecular mass oligomers possessing a stabilizing effect on membranes and anti-viral activity against tobacco mosaic virus appeared in the process of enzymatic hydrolysis of Antivir. Biological activity of 1,3;1,6-β-D-glucooligo-and polysaccharides was found to be associated with molecular mass (polymerization degree (n) not less than 14) and with presence of intralinked β-1,6-connected monosaccharide residues. Probably, decrease in molecular mass is compensated by increase in number of intralinked β-1,6-connected monosaccharide residues.

Effect of fucoidan from the brown alga Fucus evanescens on the development of infection induced by potato virus X in Datura stramonium L. leaves

We studied the influence of fucoidan from the brown alga Fucus evanescens on the development of infection induced by potato virus X (PVX) in Datura stramonium leaves. It is shown that 24 h after treatment of the leaves with fucoidan and following inoculation with PVX, the infectivity and amount of the virus in the leaves as well as the accumulation of viral particles in infected cells during early infection period were substantially less than those in the untreated control. Using ultrastructure-morphometric analysis, we established that the fucoidan treatment causes an increase in the protein-synthesizing capability of cells (nucleolus dimension and amount of both mitochondria and rough endoplasmic reticulum membranes increased). At the same time, the fucoidan treatment caused some stimulation of intracellular lytic processes that lead to destruction of virus particles and, therefore, may be considered as one of fucoidan-conditioned protective mechanisms limiting the virus accumulation in cells. Stimulation by fucoidan of the formation of PVX-specific laminated structures that may bind virus particles is another antiviral cell mechanism that limits reproduction and transportation of the virus.ZusammenfassungDie Wirkung von Fukoidan aus der Braunalge Fucus evanescens auf den Infektionsverlauf von Kartoffelvirus X (PVX) in Blättern von Datura stramonium L. wurde untersucht. Während der frühen Infektionsphase, 24 Stunden nach der Behandlung der Blätter mit Fukoidan und der folgenden Inokulation mit PVX, waren das Infektionsvermögen, der Virusgehalt in den Blättern und die Zahl der Viruspartikel in den infizierten Mesophylzellen wesentlich geringer als in den Blättern der unbehandelten Kontrolle. Durch ultrastrukturell- morphometrische Analysen wurde festgestellt, dass die Behandlung mit Fukoidan die Fähigkeit der Zellen steigert, Proteine zu synthesieren (der Nukleolus und die Zahl der Mitochondrien und Membranen des rauhen endoplasmatischen Retikulums vergrößern sich). Zugleich stimuliert die Behandlung mit Fukoidan intrazelluläre lytischen Prozesse, die Viruspartikel abbauen, weshalb diese Prozesse als durch Fukoidan ausgelöste Abwehrmachanismen betrachtet werden können, die die Akkumulation der Viren beschränken. Die Bildung von PVX-spezifischen laminaren Strukturen durch Fukoidan, die möglicherweise die Viruspartikel binden können, ist ein weiterer vom Präparat induzierter antiviraler Zellschutzmechanismus, der die Reproduktion und Ausbreitung von PVX inhibiert.

Effect of tobacco mosaic virus strains on the ultrastructure of tobacco leaf parenchymal cells

Accumulation of considerable amounts of viral particles has been demonstrated in parenchymal cells of young leaves in tobacco cultivar Samsun systemically infected with any of studied tobacco mosaic virus (TMV) strains isolated from pepper (TMV-p), tomato (TMV-t), and eggplant (TMV-e). Abnormal (swollen and thin) virions were found, which points to their destruction. Cell infection with all studied strains was accompanied by the activation of the lysosomal compartment manifested as formation of nascent dictyosomes, elements of smooth endoplasmic reticulum, cytoplasmic vacuoles, various vesicles, invaginated mitochondria, and multivesicular bodies. The studied viral strains could be arranged in the following sequence according to the degree of lysosomal compartment stimulation and induction of intracellular lytic processes mediating the destruction of viral particles and cell structures: TMV-p > TMV-e > TMV-t.

Correlation between Influence of Polysaccharides on Hydrolase Activity and Their Antiviral Effect in Tobacco Leaves

The activities of hydrolases (acid phosphatase, RNase, and proteases) in healthy and tobacco mosaic virus-infected leaves of Nicotiana tabacum L. var. Samsun, both untreated and treated with polysaccharides (PS) (1,3;1,6-β-D-glucan, fucoidan, and κ/β-carrageenan), were determined. The PS lead to substantial increase in the hydrolase level. The percentage of viral particles undergoing destructive change also increases in leaves treated with PS 24 h before infection. We suppose that the PS-mediated hydrolase activation promotes intracellular destruction of the viral particles and, thus, comprises one of the PS-induced protective mechanisms limiting intracellular viral accumulation.

Effect of fucoidan from brown alga Fucus evanescens on a formation of TMV-specific inclusions in the cells of tobacco leaves

The effect of fucoidan (1.3; 1.4-α-L-fucan), a sulfated polysaccharide from the brown alga Fucus evanescens on the formation of specific granular and tubular inclusions induced by tobacco mosaic virus (TMV) and consisted presumably of the virus-coded protein components of the viral replicase was investigated in the TMV-infected leaves of tobacco (Nicotiana tabacum L.). In four days after inoculation of the leaves with a TMV preparation (1 mg/ml), the signature of infection in a presence of fucoidan (1 mg/ml) was a preferential formation of intracellular granular inclusions, which were related to early stages of the virus reproduction. When infected leaves were not treated with fucoidan, their cells contained mainly tubular inclusions, which were presumably formed from the granular ones on the last stages of the infection process. These observations demonstrated that fucoidan delayed the development of the TMV-induced infection.

Effect of chitosan on tobacco mosaic virus (TMV) accumulation, hydrolase activity, and morphological abnormalities of the viral particles in leaves of N. tabacum L. cv. Samsun

The effect of chitosan on the development of infection caused by Tobacco mosaic virus (TMV) in leaves of Nicotiana tabacum L. cv. Samsun has been studied. It was shown that the infectivity and viral coat protein content in leaves inoculated with a mixture of TMV (2 μg/mL) and chitosan (1 mg/mL) were lower in the early period of infection (3 days after inoculation), by 63% and 66% respectively, than in leaves inoculated with TMV only. Treatment of leaves with chitosan 24 h before inoculation with TMV also caused the antiviral effects, but these were less apparent than when the virus and polysaccharide were applied simultaneously. The inhibitory effects of the agent decreased as the infection progressed. Inoculation of leaves with TMV together with chitosan considerably enhanced the activity of hydrolases (proteases, RNases) in the leaves, in comparison with leaves inoculated with TMV alone. Electron microscope assays of phosphotungstic acid (PTA)-stained suspensions from infected tobacco leaves showed that, in addition to the normal TMV particles (18 nm in diameter, 300 nm long), these suspensions contained abnormal (swollen, “thin” and “short”) virions. The highest number of abnormal virions was found in suspensions from leaves inoculated with a mixture of TMV and chitosan. Immuno-electron microscopy showed that “thin” virus particles, in contrast to the particles of normal diameter, lost the ability to bind to specific antiserum. It seems that the chitosan-induced activation of hydrolases stimulates the intracellular degradation of TMV particles and hence hydrolase activation may be considered to be one of the polysaccharide-mediated cellular defense mechanisms that limit virus accumulation in cells.

Inhibitory effect of κ/β-carrageenan from red alga Tichocarpus crinitus on the development of a potato virus X infection in leaves of Datura stramonium L.

The effect of κ/β-carrageenan from red alda Tichocarpus crinitus on the development of a potato virus X (PVX) infection in the leaves of Datura stramonium L. has been studied. The treatment of leaves with carrageenan stimulates a protein synthesis in the cells, causing an increase in the size of nucleoli and in the number of mitochondria and membranes of the rough endoplasmic reticulum. At the same time, such treatment slightly stimulates lytic processes, causing an increase in the number of smooth endoplasmic reticulum cisternae, dictyosomes, and cytoplasmic vacuoles and the formation of cytoplasmic electron-transparent zones. The carrageenan-induced stimulation of lytic processes results in the destruction of viral particles and can be considered as one of the defense mechanisms, preventing the intracellular accumulation of virus. The carrageenan-stimulated formation of PVX-specific laminar structures, able to bind viral particles and, therefore, prevent their intracellular translocation and reproduction, represents another carrageenan-induced mechanism of the antiviral defense in plant cells.