THE EFFECTS OF VARYING THE WATER SUPPLY OF PLANTS ON THEIR SUSCEPTIBILITY TO INFECTION WITH VIRUSES

Increasing the amount of water supplied to plants before they were inoculated with viruses greatly increased their susceptibility to infection; plants that received unlimited water produced 10 or more times as many local lesions as plants that received only enough to prevent wilting. Susceptibility was increased throughout the year, but the full response occurred in 2 weeks in winter and 4 weeks in summer. Plants that received unlimited water for the 2 weeks immediately preceding inoculation were no more susceptible than those that received it during the previous 2 weeks, although the external appearance of the plants differed at the time of inoculation. Varying water supply after inoculation did not affect the numbers of lesions.
The differences in susceptibility to infection produced by differential watering were decreased, but not abolished, by growing plants under shade or by incorporating a diatomaceous earth in the inoculum.
Increasing water produced plants with larger and more succulent leaves; the cuticular layer was thinner, and the palisade tissue was less regularly arranged than in the plants kept dry. The increased susceptibility caused by an abundant water supply may be at least partly due to these structural differences, which allow the leaf to be damaged more easily when rubbed with inocula and so present more entry points for virus particles.     

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.     

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.     

Patterns of Stomatal Behaviour, Transpiration, and CO2 Exchange in Pea Following Infection by Powdery Mildew (Erysiphe pisi)

A comparison was made of stomatal behaviour, and related phenomena,between leaves of garden pea (Pisum sativum cv. Feltham First)inoculated with powdery mildew fungus (Erysiphe pisi) and uninfectedleaves on healthy plants. Twenty four hours after inoculation,stomata opened more widely in the light in infected leaves thanin healthy leaves. Thereafter, stomatal opening was progressivelyreduced by infection and stomata failed to close completelyin the dark until, 7 d after inoculation, all movements ceasedand stomata remained partly open. Transpiration in the lightfollowed closely the pattem of stomatal opening and, after anearly increase compared with healthy controls, was progressivelyreduced by infection. Evidence is presented that transpirationfrom the fungus was less than the reduction in transpiraationfrom the leaf which was caused when development of the myceliumincreased the boundary layer resistance of the leaf. Seven daysafter inoculation, transpiration in the dark was greater frominfected leaves than from healthy leaves because of partly openstomata in the dark. Net photosynthesis in infected leaves was reduced within 24h of inoculation to a level below that found in healthy leavesand thereafter it declined progressively. The initial reductionwas due to a transient increase in photorespiration, for whenthe glycolate pathway was inhibited by a 2% O₂ concentrationthere was no difference between the (gross) photosynthetic ratesof healthy and infected leaves. Changes in photorespirationrate were confirmed from the interpretation of the CO₂ burston darkening. Reduced stomatal opening was a contributory causeof the reduction in net photosynthesis in the later stages ofinfection. Since the rate of gross photosynthesis, but not therate of photorespiration, of infected plants fell below thatof healthy plants, and infected plants had a higher rate ofrelease of CO₂ in the dark than healthy plants from the thirdday after inoculation onwards, infected plants consume an increasinglygreater proportion of their photosynthate in respiratory processesthan do healthy plants. The CO₂ compensation point of infectedplants increased at every time of sampling after inoculation.     

FACTORS AFFECTING THE PRODUCTION OF LOCAL LESIONS BY PLANT VIRUSES

Beans inoculated with tobacco necrosis virus were kept in the dark at different temperatures for 1 hr. before and 1 hr. after inoculation; in this experiment the number of lesions increased with temperature over the range 55–82° F.
The effect of 30 min. periods of darkness before or after inoculation depended on the time of day, the number of local lesions usually being decreased. Prolonging the night period before inoculation sometimes increased the number of lesions.
Light appeared to be more important than temperature in controlling the daily variation in susceptibility. However, in a test over a 30 hr. period this variation continued even when plants were placed under constant conditions before and after inoculation.
When plants that had been kept in the dark were exposed to light of about 800 f.c. intensity for 1 min. immediately before inoculation the number of local lesions was doubled.     

SOME EFFECTS OF NITROGEN SUPPLY ON THE INFECTION OF TOMATO PLANTS WITH TOMATO SPOTTED WILT VIRUS

The susceptibility of tomato plants to systemic infection by tomato spotted wilt virus was increased by increasing nitrogen supply to levels above that optimal for growth. The virus content, estimated by local-lesions counts, was also raised by increasing nitrogen. The period between inoculation and the appearance of systemic symptoms was decreased by increasing nitrogen to a point slightly greater than the optimal level for growth, but increased by additional applications.
Infected plants receiving more nitrate or ammonium compounds than were needed for optimal growth showed abnormal leaf symptoms and no bronzing. N, P and Mg analyses showed that these symptoms were related primarily to nitrogen content. Such leaves contained more virus than bronzed leaves.     

THE EFFECT OF TIME OF APPLICATION OF NITROGENOUS FERTILIZER ON POWDERY MILDEW OF WINTER WHEAT

Applying nitrogenous fertilizer (N) to wheat plants of different ages affected the incidence of powdery mildew, Erysiphe graminis DC, differently. When N was applied before the flag leaf had emerged, the infection-index (number of pustules per 100 sq.cm. of leaf blade) increased to a maximum and then declined. If N was applied after the flag leaf had emerged, the infection-index increased steadily without reaching a peak, and the increased susceptibility was not associated with an increased relative leaf growth rate as when N was applied earlier.
The integral of the number of pustules per unit area with time, and the number of perithecia on the leaves of the main stem, both increased with increasing delay in the application of N. Plants given N in either April or May had at least three times as many pustules as those given N in January. The time when N was applied did not affect the date when perithecia appeared.
After adding N to nitrogen-deficient plants, the already mature leaves which had resisted mildew infection, became susceptible. This change is not associated with changes in the epidermal wall.     

Oscillations in stomatal conductance of hybrid poplar leaves in the light and dark

Cycling of stomatal conductance in three hybrid poplar ( Populus sp.) cultivars was observed under a variety of conditions. Illumination of plants kept previously in the dark induced very large oscillations with a period of about 40 min and large oscillations with a shorter period (< 10 min) were superimposed on the longer cycles. During these oscillations, large changes in conductance could occur very rapidly (1.0 cm s⁻¹ in 3 min). Plants in constant light also displayed both long and short term cycles in conductance, but these were smaller in amplitude than those induced by sudden illumination. Stomatal oscillations were also observed in darkness and after darkening of previously illuminated plants. These oscillations had shorter (< 30 min) and less regular periods than those observed in the light. Such cycling in the dark is rare. Cycling of the two leaf surfaces was sometimes in synchrony in the light, and more so after a perturbation. Little synchrony between the two surfaces was observed in the dark. Stomatal movements of different leaves on a plant were usually relatively independent. Transient stomatal opening occurred following leaf excision in the light or dark, and often after sudden darkening of intact leaves. Also, stomata of intact leaves sometimes transiently closed following illumination.     

THE EFFECTS OF STREPTOMYCIN AND TERRAMYCIN, SINGLY AND IN COMBINATION, ON THE LEAF BLIGHT DISEASE OF MAIZE CAUSED BY BACTERIUM CAROTOVORUUM F. ZEAE SABET

The growth in vitro of Bacterium carotovorum f. zeae Sabet, the cause of the root and stalk rot and leaf blight disease of maize, was inhibited by dihydrostreptiomycin sulphate and by Terramycin (oxytetracycline). Joint applications showed a synergistic action, and the bacterium was most susceptible to a 1:1 mixture of the two antibiotics. This mixture is effective in checking the development of resistant strains which develop rapidly after treatment with streptomycin alone.
Roots of maize seedlings in water cultures, and leaves of older plants readily absorb both antibiotics. High water content of soil favoured absorption; sunlight had no effect. Absorption from leaf applications first increased with higher concentration and frequency of spraying, but after eight daily applications of streptomycin toxic symptoms appeared and there was evidence that the antibiotic was inactivated.
Antibiotic treatment of maize leaves shortly before inoculation with B. carotovorum f. zeae reduced the incidence and severity of leaf blight. The therapeutic effect extends at first both downward and upward but later only upward. Treatment of the leaves did not induce resistance to infection in the stems and roots.     

THE INDUCTION OF LEAF TUMORS BY AGROBACTERIUM TUMEFACIENS

Using carborundum as an abrasive and light rubbing with a culture of Agrobacterium tumefaciens, leaves of various species of bean and tobacco develop tumors on the leaf lamina. The induction of these tumors requires wounding, the presence of a virulent strain of the bacterium and is due to the bacterium, not substances released into the bacterial culture medium during growth. Observations of the histology and cytology of these tumors on the primary leaves of pinto bean show no significant differences from the more commonly studied stem tumors. The tumors on pinto beans first appear as chlorotic nests of dividing cells which gradually accumulate chlorophyll, eventually becoming dark green in color as opposed to the surrounding leaf tissue which is completely chlorotic at this stage. Tumor development is enhanced by a dark period following inoculation while growth of the leaf is essentially stopped. The tumors thus exhibit a pattern of growth and development independent of that of the normal leaf. The number of tumors obtained on pinto bean leaves was found to depend on the concentration of bacteria in the inoculum and on the age of the plants. A sharp peak in response was observed at about 7 days from planting. Best results were obtained by adding the bacterium at the time of wounding. The tumors were shown to differ from IAA-induced leaf proliferations with respect to their point of origin on the leaf, morphology, physiology and development.     

Stomatal response of hybrid poplar to incident light, sudden darkening and leaf excision

Responses of abaxial and adaxial stomata of Populus trichocarpa Torr. & Gray. × P. deltoides Bartr. (ex Marsh.) cv. Unal to incident light, sudden darkening and leaf excision in the light and in the dark were studied on 5-year-old trees in the field using diffusion porometry. Stomatal closure in the dark was found to be incomplete in most cases studies. Stomata closed after leaf excision in the dark within 90 min. Stomatal closure after darkening of an entire tree or an entire branch (white the rest of the tree was in the light) was slower, and complete stomatal closure was noticed only for adaxial stomata after 3 h. Adaxial stomata were more reactive and sensitive than abaxial stomata to sudden darkening and leaf excision in the light and the dark. In all treatments, stomatal response was more responsive in mature leaves than in young, still expanding leaves.     

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 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.     

Photosynthesis and predisposition of plants to infection with certain viruses

The effects on susceptibility to infection with certain viruses of subjecting plants to various periods of darkness or reduced illumination before and after inoculation were tested. The viruses and hosts used were a tobacco necrosis virus in French bean and tobacco; tomato aucuba mosaic virus in tobacco; and tobacco mosaic and tomato bushy stunt viruses in Nicotiana glutinosa . All the virus-host combinations give necrotic local lesions, and susceptibility was measured by local lesion counts. Susceptibility was consistently increased by pre-inoculation treatments of host plants, whereas post-inoculation treatments had relatively little effect, but most often decreased susceptibility.
Short periods in the dark produced similar responses to longer periods in shade, but the different plants varied in their response to, and tolerance of, darkness. The maximum number of lesions was usually obtained with bean plants kept for only 24 hr. in the dark before inoculation, but with tobacco plants susceptibility increased with increasing time in the dark up to 5 days.
It is suggested that the successful establishment of infection occurs in two stages, the first of which is affected by. the accumulation of photosynthetic products. Whether these products confer resistance by increasing cell turgor or by reacting specifically with virus particles is unknown, but sap from plants in the light possesses no greater virus-inhibiting power than sap from plants kept in the dark.     

PHYSIOLOGICAL STUDIES ON THE VERTICILLIUM WILT DISEASE OF TOMATO

The water loss per unit leaf area of tomato plants was decreased after inoculation with Verticillium albo-atrum. When diseased plants began to wilt water loss temporarily increased, but then rapidly decreased to become less than that of healthy plants grown under conditions of adequate or restricted water supply.
The transpiration of excised leaves from plants grown with a restricted water supply was reduced, but not so severely as that of comparable leaves from infected plants. Water loss from leaves on infected plants was reduced irrespective of any blocking of the petiolar xylem.
The rate of water loss from turgid leaf disks on mannitol solutions, and the rate of water uptake of leaf disks on water was similar for disks cut from wilting or turgid leaves of diseased plants or healthy plants grown with an adequate or restricted water supply.
Disease or poor water supply reduced leaf growth but had no effect on the rate of leaf initiation. Although the density of stomata was higher on leaves of diseased plants the stomatal area was less than on healthy plants.
The resistance to water flow in diseased stems was high and was correlated with vessel blockage. About half the blocked vessels contained hyphae. The severity and localization of symptoms in inoculated plants growing on susceptible or resistant rootstocks was directly related to the extent of invasion by the pathogen and to vessel blockage.
Experiments on the wilting activity of cell-free filtrates from cultures of the pathogen in vitro indicated that it produced a stable substance, not an enzyme, that caused wilting in cut shoots by blocking the end of the stem. It is suggested that an increasing internal water shortage causes major symptoms of disease.     

Some effects of darkness and partial defoliation on the resistance of sugar beet to downy mildew

Seedlings of two susceptible sugar-beet stocks were grown either in daylight or in darkness for 24 h before or after inoculation with Peronospora farinosa f.sp. betae. Post-inoculation darkness did not significantly affect the percentage of inoculated plants that became infected but increased the percentage of plants on which the fungus sporulated in both stocks. The effects on sporulation of darkness before inoculation were more variable than those of post-inoculation darkness. In the same stocks removal of all leaves, except those in the bud, before inoculation increased the percentage both of plants which became infected and of those on which P. farinosa sporulated. Infection and sporulation were not significantly affected by defoliation in a more resistant stock. Defoliation or darkness may have affected the concentration of certain sugars or amino acids in the bud leaves thereby changing the expression of polygenically-controlled resistance to downy mildew.     

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 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.     

AN ANALYSIS OF THE GROWTH RESPONSE OF YOUNG TOMATO PLANTS TO INFECTION BY VERTICILLIUM ALBO-ATRUM

Infection of seedling tomatoes with Verticillium albo-atrum checked growth but did not result immediately in leaf yellowing. Localized wilting occurred in some plants 2 weeks after the check to growth was evident. 8 weeks after inoculation, dry weights of leaf, stem and root were decreased by 72, 70, and 65% respectively.
Of the growth attributes studied, leaf area was most reduced by infection and this was due to a failure of the leaves to expand rather than to a fall in the rate of leaf production. Neither water nor nitrogen appeared to be limiting factors in this respect. The water content of infected leaves was not reduced until 6 weeks after inoculation, when leaf yellowing and necrosis had also appeared. The percentage N contents of stem, root and leaf of infected plants exceeded those of the healthy controls 24 days after inoculation. N uptake was not seriously impaired until 21 days later.
The photosynthetic efficiency of the green leaves of infected plants was reduced. The mean values for net assimilation rates were: Healthy 0.47 and infected 0.39 g./dm.²/week.
Plants, in which two-thirds of the root system had been killed by crushing, were placed in contact with mycelium in soil. This initial root injury did not significantly affect the growth of infected plants.
The data accord with a toxin theory of damage to infected plants, but the slow development of chlorosis and wilting symptoms in the young plants suggested a greater tolerance to the toxin than is found in older plants.     

CYCLING OF STOMATAL APERTURE IN LEAVES OF PLANTS WITH CRASSULACEAN ACID METABOLISM UNDER CONSTANT CONDITIONS

When leaves of plants with C₃ metabolism are detached and held in darkness, they senesce and the stomata close. Because the relation of senescence and stomatal closure is very close, if not actually causal, the question arose as to whether in the leaves of plants with Crassulacean acid metabolism whose stomata open at night the relationship to senescence would be reversed. Detached leaves of four species of Hoya, floated on water in constant darkness or constant light, were found to show no large differences in stomatal aperture (measured as diffusion resistance) between those in the light or dark, but the aperture changed in a regular circadian rhythm. In some leaves the rhythm was simple, in others the peak showed small secondary peaks, but in all cases the values were nearly the same in the light as in the dark, throughout the cycle. Previous culture of the intact plants under normal day/night conditions gave results similar to those with plants that had had prolonged culture under constant light or darkness. In those cases when the stomata were more open in the dark, the chlorophyll content was greater than when the stomata were more open in the light; but when they were more open in the light, the chlorophyll content showed little difference between light and dark. When the leaves had only their petioles in water they showed greater senescence in the light than in the dark, and the stomata were more tightly closed in the light, especially at the apical ends. All four species of Hoya gave similar results. We deduce that senescence of these leaves is modified by stomatal aperture, and generally in the same direction as in C₃ leaves, but that in continuous light or darkness the primary control over the aperture is the endogenous cycle.