THE EFFECT OF INFECTION WITH TOBACCO MOSAIC VIRUS ON THE RESPIRATION OF TOBACCO LEAVES OF VARYING AGES IN THE PERIOD BETWEEN INOCULATION AND SYSTEMIC INFECTION

Leaves of tobacco plants inoculated with tobacco mosaic virus were divided into three groups: ( a ) inoculated leaves; ( b ) younger non-inoculated leaves present at the time of inoculation; ( c ) leaves formed since inoculation. The respiration rate of each group was compared with that of similar leaves from healthy plants. The respiration rate of inoculated leaves was increased by a constant amount for 3 weeks after inoculation, when it decreased. The respiration rate of group ( b ) leaves was not affected at any time, and that of group ( c ) leaves was decreased by 10% when they showed symptoms. The increased respiration in the inoculated leaves occurred too soon to reflect virus formation, and it is suggested that it reflects an initial change in infected cells preparatory to virus synthesis. The subsequent decrease in respiration may be due to the accumulation of virus which does not contribute to the total leaf respiration.     

THE EFFECT OF INFECTION WITH TOBACCO ETCH VIRUS ON THE RATES OF RESPIRATION AND PHOTOSYNTHESIS OF TOBACCO LEAVES

Unlike tobacco mosaic virus, which increases the respiration of tobacco leaves within an hour of their being inoculated, a virulent strain of tobacco etch virus did not change respiration rates until leaves showed external symptoms. The respiration rates of inoculated or systemically infected leaves with symptoms rose to 40% above that of healthy leaves, three times the increase produced by tobacco mosaic virus. The increased respiration rate occurred at all times of the year and was maintained through the life of the leaves.
Leaves infected with tobacco etch virus and showing symptoms had a photo-synthetic rate 20% lower than that of healthy leaves.     

THE EFFECTS OF INFECTION WITH TOBACCO MOSAIC VIRUS ON THE PHOTOSYNTHESIS OF TOBACCO LEAVES

The rate of photosynthesis of tobacco leaves infected with the Rothamsted type culture of tobacco mosaic virus was lower than that of comparable healthy tobacco leaves. The lower rate was inferred from Net Assimilation Rates of whole plants and confirmed by direct comparisons of photosynthetic rates of inoculated and healthy leaves. The effect began within 1 hr. of inoculation. It was not caused by an effect of the virus on the stomata, and inactivated virus inoculum did not change the rates. The results indicate either a more rapid movement of virus from the epidermis into the chlorenchyma than has been previously recorded or an effect of virus infection at a site distant from the cells containing virus.     

THE RESPIRATION OF TOBACCO LEAVES IN THE 20-HOUR PERIOD FOLLOWING INOCULATION WITH TOBACCO MOSAIC VIRUS

The effect of infection with tobacco mosaic virus on the respiration rates of detached tobacco leaves in the period immediately after inoculation differed in plants grown at different times of the year. During winter, infection increased respiration rates, and in summer decreased them. In winter-grown plants, increasing the light intensity during the period before inoculation decreased respiration rates after infection. Extending the day length for winter-grown plants did not alter the effect of infection on respiration. Respiration rates began to change in less than 1 hr. after inoculation and are unlikely to be associated with the formation of new virus.     

SOME EFFECTS OF VIRUS INFECTION ON LEAF WATER CONTENTS OF NICOTIANA SPECIES

Infection with tobacco mosaic virus decreases the water content which detached tobacco leaves attain when kept for 20 hr. in conditions of minimum water stress, and does so more when the plants are kept in light before inoculation than when they are kept in darkness. No such effects of infection during the first day after inoculation were obtained with tobacco leaves infected with either tobacco etch virus or potato virus X , or with Nicotiana glutinosa leaves infected with tobacco mosaic virus. These results, like those showing early effects of TMV on respiration and photosynthesis of tobacco leaves, suggest that inoculation with TMV affects deeper leaf tissues than the epidermis earlier in tobacco leaves than in other leaves, and earlier than other viruses in tobacco leaves.     

THE EFFECT OF INFECTION WITH BEET YELLOWS AND BEET MOSAIC VIRUSES ON THE CARBOHYDRATE CONTENT OF SUGAR-BEET LEAVES, AND ON TRANSLOCATION

The loss of total carbohydrate (sugars and starch) per cent of residual dry matter (dry matter less total carbohydrate) during a period of darkness from leaves of sugar-beet plants infected with yellows virus was as great as that from the leaves of healthy plants. The conclusion of previous workers, based on the results of the Sachs iodine test for starch and the occurrence of 'phloem gummosis' in infected plants, that starch accumulates in infected leaves because translocation is prevented by blockage of the sieve-tubes, is therefore incorrect.
Older leaves of infected plants had a higher content of reducing sugars and sucrose, and usually but not invariably of starch, both at the beginning and end of the dark period, than comparable leaves of healthy plants. By far the greater part of the increase was in reducing sugars. In leaves taken in late September from infected plants growing in the field, 20 % or more of the total dry matter was present as reducing sugars. The reducing sugars in both healthy and yellows-infected leaves were shown by paper chromatography to be glucose and fructose in approximately equal amounts.
Accumulation of carbohydrate in infected leaves is probably not a passive consequence of differences in carbohydrate production, distribution and utilization, but is attributable to changes in the physiology of the cells of the leaf.
The carbohydrate content of sugar-beet leaves was little affected by infection with beet mosaic virus.
Yellows-infected leaves had a lower water content per cent of fresh weight than healthy leaves. This was accounted for by the higher carbohydrate content of infected leaves, for the ratio of water: residual dry matter was not affected by infection or was slightly reduced. This implies that hydration was independent of carbohydrate content.     

PHOTOSYNTHESIS AND RESPIRATION RATES OF LEAVES OF NICOTIANA GLUTINOSA INFECTED WITH TOBACCO MOSAIC VIRUS AND OF N. TABACUM INFECTED WITH POTATO VIRUS X

The rates of respiration and of photosynthesis of tobacco leaves infected with potato virus X were not affected until the leaves showed symptoms; the respiration rate was then increased by more than 30% and the photosynthesis rate decreased by 20%. When local lesions appeared on the leaves of Nicotiana glutinosa infected with tobacco mosaic virus, but not before, the respiration rate was increased by an amount, up to 30%, that varied with the number of lesions. The photosynthesis rate was decreased by 20%, but there was no effect on photosynthesis or respiration until symptoms appeared. These results differ from those previously reported for tobacco leaves infected with tobacco mosaic virus, in which both respiration and photosynthesis were affected within 1 hr. of inoculation. The validity of extrapolating arguments based on the results obtained with other combinations to this commonly used combination and vice-versa is questioned.     

Chemical suppression of the symptoms of two virus diseases

Carbendazim applied at the rate of 2 g per plant to the roots of tobacco (Nicotiana tabacum cv. White Burley) plants before infection with tobacco mosaic virus (TMV) caused very considerable reduction in the severity of disease symptoms in systemically infected leaves but did not affect their virus content. Leaves of untreated, infected plants had a greatly reduced chlorophyll content 100 days after infection whereas the chlorophyll content of leaves of infected plants treated with carbendazim was similar to that of normal uninfected leaves. Carbendazim had no effect on the infectivity of TMV in vitro or on the local lesion reaction of N. glutinosa plants when inoculated with TMV. Carbendazim was applied to lettuce cv. Cobham Green at a total rate of o-i g per plant before and after they were infected with beet western yellows virus and the plants were then grown on in the field. At harvest time (50 days after infection) almost all the treated virus-infected plants were of a normal green appearance, whereas the untreated controls were almost all very severely yellowed and unmarketable.     

SOME EFFECTS OF TANNIC ACID AND OF LEAF EXTRACTS WHICH CONTAIN TANNINS ON THE INFECTIVITY OF TOBACCO MOSAIC AND TOBACCO NECROSIS VIRUSES

The inhibition of infection by tobacco necrosis and tobacco mosaic viruses by tannic acid, and by extracts of raspberry and strawberry leaves, was associated with the precipitation of the viruses. Precipitation and inhibition were reversible, and infective virus was obtained from the precipitate formed between the viruses and tannins. Infectivity was fully restored by diluting mixtures of virus and tannin adequately and partially restored by adding alumina or nicotine sulphate.
Viruses and tannins are thought to form non-infective complexes, in which the virus and tannin components are held together by co-ordinate linkages or hydrogen bonds.
Macerating tobacco leaves infected with tobacco mosaic virus together with raspberry leaves greatly decreased the infectivity of the extracts; adding nicotine sulphate to the mixture of leaves before it was ground increased the infectivity, even though nicotine sulphate alone decreases the infectivity of tobacco mosaic virus. Even in the presence of nicotine sulphate, much of the virus was precipitated by substances from the raspberry leaves.
Extracts of roots of Fragaria vesca plants, infected with a tobacco necrosis virus, were more infective when made by macerating the roots with four times their weight of buffer at pH 8 than when made without buffer. Various methods are suggested for facilitating the transmission of viruses from plants that contain tannin.     

THE PREPARATION AND USE OF TOBACCO MOSAIC VIRUS CONTAINING RADIOACTIVE PHOSPHORUS

Normal and tobacco mosaic-diseased Turkish tobacco plants were grown in sand for a period of several weeks, during which they were fed daily a complete nutrient solution to which had been added disodium phosphate containing radioactive phosphorus. Determinations were made of the distribution of radioactive phosphorus in different fractions such as the wash from the sand and roots, the press cake obtained on pressing the juice from the plants, the protein and protein-free portions of the supernatant liquids obtained on ultracentrifugation of the juices, and the purified tobacco mosaic virus isolated from the diseased plants. Chemical analyses as well as radiographs of the normal and diseased leaves indicated that they contained the same amount of phosphorus. Approximately 30 per cent of the radioactive phosphorus absorbed by the diseased plants was found to be combined with the purified tobacco mosaic virus that was isolated from these plants. Following the inoculation of purified tobacco mosaic virus possessing high radioactivity to normal Turkish tobacco plants, most of the radioactivity was found to be associated with non-virus components of which about 40 per cent was in the inoculated and 60 per cent in the uninoculated portions of the plants. Although a small amount of radioactive virus was isolated from the uninoculated portions of the plants, it was impossible, because of a number of complicating factors which have been discussed, to draw from the results any reliable conclusions regarding the mode of reproduction of tobacco mosaic virus.     

THE INFLUENCE OF LIGHT INTENSITY ON THE SUSCEPTIBILITY OF PLANTS TO CERTAIN VIRUSES

Reducing the light intensity under which plants were grown in summer to one-third increased their susceptibility to infection with tobacco necrosis, tomato bushy stunt, tobacco mosaic and tomato aucuba mosaic viruses. With the first two viruses shading increased the average number of local lesions per leaf by more than ten times and by more than five times with the second two.
Reducing the light intensity increased the virus content of sap from leaves inoculated with Rothamsted tobacco necrosis virus by as much as twenty times. As it also reduced the total solid content of sap by about one-half, purification was greatly facilitated; crystalline preparations of the virus were readily made from shaded plants but not from unshaded controls.
Reducing the light intensity also increased the virus content of systemically infected leaves; the greatest effect was with tomato bushy stunt virus with which increases of up to ten times were obtained, but with tobacco mosaic and aucuba mosaic viruses there were also significant increases.
The importance of controlled illumination in raising plants for virus work and the possible mechanisms responsible for the variations in susceptibility are discussed.     

Tobacco-mosaic-virus-induced increase in abscisic-acid concentration in tobacco leaves:

The concentrations of free and bound abscisic acid (ABA and the presumed ABA glucose ester) increased three- to fourfold in leaves of White Burley tobacco (Nicotiana tabacum L.) systemically infected with tobacco mosaic virus. Infected leaves developed a distinct mosaic of light-green and dark-green areas. The largest increases in both free and bound ABA occurred in dark-green areas. In contrast, virus accumulated to a much higher concentration in light-green tissue. Free ABA in healthy leaves was contained predominantly within the chloroplasts while the majority of bound ABA was present in non-chloroplastic fractions. Chloroplasts from light-green or dark-green tissues were able to increase stromal pH on illumination by an amount similar to chloroplasts from healthy leaf. It is unlikely therefore that any virus-induced diminution of pH gradient is responsible for increased ABA accumulation. Tobacco mosaic virus infection had little effect on free ABA concentration in chloroplasts; the virus-induced increase in free ABA occurred predominantly out-side the chloroplast. The proportional distribution of bound ABA in the cell was not changed by infection. Treatment of healthy plants with ABA or water stress increased chlorophyll concentration by an amount similar to that induced by infection in dark-green areas of leaf. A role for increased ABA concentration in the development of mosaic symptoms is suggested.Abbreviations ABA abscisic acid - TMV tobacco mosaic virus     

THE INOCULATION OF TOBACCO CALLUS TISSUE WITH TOBACCO MOSAIC VIRUS

Although cultures of normal and conditioned tobacco callus tissue occasionally became infected when dilute solutions of tobacco mosaic virus was poured over them, injuries were usually required, and the number of infections depended on the type and number of injuries. Tissues infected by superficial injuries usually became virus-free after subculturing, whereas those infected by needle-prick remained infected permanently. Although no plasmodesmata were found joining cells in the tissue cultures, tobacco mosaic virus moved between them at a rate of about 1 mm. per week, approximately the same rate as it moves through cells of the leaf parenchyma.     

SOME EFFECTS OF HOST-PLANT NUTRITION ON THE MULTIPLICATION OF VIRUSES

The amounts of tobacco mosaic virus present in systemically infected tobacco plants varied greatly with the mineral nutrition of the plants and were related to the effects on plant growth. With plants in soil, supplements of phosphorus produced the greatest increases in plant size, in virus concentration of expressed sap, and in total virus per plant; nitrogen increased plant size only when phosphorus was also added, and only then increased virus concentration and total virus per plant. Combined supplements of phosphorus and nitrogen doubled the virus concentration of sap and increased the total virus per plant by factors up to forty. Potassium slightly reduced the virus concentration of sap, though it usually increased plant size and total virus per plant. From all plants, only about one-third of the virus contained in leaves was present in sap. Virus production seemed to occur at the expense of normal plant proteins, and the ratio of virus to other nitrogenous materials was highest in plants receiving a supplement of phosphorus but not of nitrogen.
The effects of host nutrition on the production of virus in inoculated leaves resembled those in systemically infected leaves, but were more variable.
No evidence was obtained, with plants grown in soil or sand, that host nutrition had any consistent effect on the intrinsic infectivity of tobacco mosaic virus.
The concentration of virus in sap from potato plants systemically infected with two strains of potato virus X was not consistently affected by fertilizers; the chief effect of host nutrition on virus production was indirect by altering plant size.     

EFFECTS OF DARKNESS ON THE CONSTITUTION OF TOBACCO LEAVES AND SUSCEPTIBILITY TO VIRUS INFECTION

An attempt was made to find the causes of increased susceptibility to virus infection when tobacco plants are kept in the dark before inoculation. The changes in certain nitrogen fractions, viz. insoluble-N, amino-N, amide-N, ammonia-N and nitrate-N, and in dry matter and water content were followed in tobacco plants subjected to a period of darkness before inoculation with tobacco aucuba mosaic virus. Only nitrate-N was strongly correlated with the susceptibility to infection, but the evidence suggests that the correlation is indirect and not causal.
Dry matter and water content, determined either as dry matter percentage of fresh weight or measured separately on a leaf-disk basis Ivere found to vary directly with variation in susceptibility.     

Effect of yeast CTA1 gene expression on response of tobacco plants to tobacco mosaic virus infection

The response of tobacco (Nicotiana tabacum L. cv Xanthi-nc) plants with elevated catalase activity was studied after infection by tobacco mosaic virus (TMV). These plants contain the yeast (Saccharomyces cerevisiae) peroxisomal catalase gene CTA1 under the control of the cauliflower mosaic virus 35S promoter. The transgenic lines exhibited 2- to 4-fold higher total in vitro catalase activity than untransformed control plants under normal growth conditions. Cellular localization of the CTA1 protein was established using immunocytochemical analysis. Gold particles were detected mainly inside peroxisomes, whereas no significant labeling was detected in other cellular compartments or in the intercellular space. The physiological state of the transgenic plants was evaluated in respect to growth rate, general appearance, carbohydrate content, and dry weight. No significant differences were recorded in comparison with non-transgenic tobacco plants. The 3,3'-diaminobenzidine-stain method was applied to visualize hydrogen peroxide (H(2)O(2)) in the TMV infected tissue. Presence of H(2)O(2) could be detected around necrotic lesions caused by TMV infection in non-transgenic plants but to a much lesser extent in the CTA1 transgenic plants. In addition, the size of necrotic lesions was significantly bigger in the infected leaves of the transgenic plants. Changes in the distribution of H(2)O(2) and in lesion formation were not reflected by changes in salicylic acid production. In contrast to the local response, the systemic response in upper noninoculated leaves of both CTA1 transgenic and control plants was similar. This suggests that increased cellular catalase activity influences local but not systemic response to TMV infection.     

HEAT-THERAPY OF VIRUS-INFECTED PLANTS

Virus-free plants were produced from parents systemically infected with the following five viruses: tomato bushy stunt, carnation ring spot, cucumber mosaic, tomato aspermy and Abutilon variegation. The leaves formed while the infected plants were kept at 36°C. were free from symptoms, and test plants inoculated from these remained uninfected. When cuttings were taken from the infected plants at the end of the treatment most grew into healthy plants. The treated plants themselves usually developed symptoms after varying lengths of time at 20°C, but some that before treatment were infected with tomato aspermy, cucumber mosaic or Abutilon variegation viruses, remained permanently healthy.
The same method failed to cure plants infected with tomato spotted wilt, potato virus X and tobacco mosaic virus, although it decreased their virus content. Heat-therapy seems not to be correlated with the thermal inactivation end point of the virus in vitro.     

EFFECTS OF TOBACCO MOSAIC VIRUS ON EARLY LEAF DEVELOPMENT IN TOBACCO

Tepfer , Sanford S. (U. Oregon, Eugene.), and Meyer Chessin . Effects of tobacco mosaic virus on early leaf development in tobacco. Amer. Jour. Bot. 46(7): 496–509. Illus. 1959.—At certain stages of infection, tobacco mosaic virus causes the formation of highly abnormal tobacco leaves classified here as narrow-bladed leaves and “shoestring” leaves. The development and anatomy of these types are described. The “shoestring” leaf in its extreme form is entirely radial in symmetry, with no vestige of a lamina. This suppression of dorsiventrality is expressed to a lesser degree in narrow-bladed leaves and in transitional forms. The lack of or reduction of dorsiventrality results directly from the absence of or reduced activity of the marginal meristems of the leaf primordium. There is a general reduction in meristematic activity in the primordium that causes reduced length as well as reduced laminal development.     

PRIMULA OBCONICA, A CARRIER OF TOBACCO NECROSIS VIRUSES

A tobacco necrosis virus has been isolated from the leaves and flowers of naturally infected Primula obconica plants. Although the virus produces no necrotic symptoms, it is not distributed uniformly through primulas, but occurs only in isolated regions, most of the tissues being apparently virus-free.
When inoculated to healthy primulas, three tobacco necrosis viruses were found to behave similarly. They all enter and multiply locally, but produce no symptoms; movement from the inoculated areas occurs only rarely and then does not cause a full systemic infection but only further localized infections. Multiplication of the viruses in primula is slower than in tobacco or French bean, which react necrotically.
The uncertainties in interpreting results of tests for tobacco necrosis viruses are described and possible explanations of natural infections are discussed.