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.     

THE EFFECT OF TEMPERATURE ON INFECTION WITH STRAINS OF CUCUMBER MOSAIC VIRUS

Cucumber mosaic virus strains differed in their ability to multiply in plants at 37° C. Some strains multiplied in inoculated leaves and produced systemic symptoms in plants at this temperature; plants systemically infected with one such strain remained infected after prolonged treatment at 37° C. Other strains did not appear to multiply in inoculated leaves at 37° C. and heat treatment was successful in freeing plants from infection with these. Tests with one strain of each type showed both to be rapidly inactivated in expressed sap at 37° C.
Strains of cucumber mosaic virus forming small necrotic local lesions in leaves of french bean var. Canadian Wonder, produced many fewer lesions in plants kept after inoculation at 25° C. for 24 hr. and then at 15° C. than in plants kept continuously at the lower temperature.     

VIRUSES OF COWPEA, VIGNA UNGUICULATA L. (WALP.), IN NIGERIA

The cowpea strain of tobacco mosaic virus was isolated from a range of leguminous hosts at Ibadan, but was rare in cultivated crops. Systemic symptoms in species infected experimentally are described.
A new virus of cowpea was also found in Nigeria. The physical properties (thermal inactivation point 56° C., dilution end-point 1/50,000 and longevity in vitro 4 days at 25° C.) differ from those for cowpea viruses reported elsewhere and the name cowpea yellow mosaic virus is proposed. This virus produces local lesions in French bean ( Phaseolus vulgaris L.) and local and systemic lesions in Bengal bean ( Mucuna aterrima Holland), but does not infect other leguminous hosts. The virus was purified and an antiserum prepared against it.
Both viruses are transmitted by a beetle ( Ootheca mutabilis Sahlb.) which loses infectivity within 48 hr. of leaving plants infected with either or both viruses.     

SOME EFFECTS OF CHANGING TEMPERATURE AND OF VIRUS INHIBITORS ON INFECTION BY CUCUMBER MOSAIC VIRUS

Whereas the spinach strain of cucumber mosaic virus fails to multiply and cause symptoms in tobacco plants kept above 30° C., the yellow strain infects at 36° C. and causes more severe symptoms than at 20° C. Increasing the temperature up to 28° C. increases the initial rate at which the spinach strain multiplies, but the virus later reaches much higher concentrations in leaves at lower temperatures, presumably because it is rapidly inactivated at 28° C. Exposing inoculated plants to 36° C. for 6 hr. decreases the number of infections by the spinach strain when the exposure starts within 6 hr. of inoculation, but not afterwards.
Pancreatic ribonuclease inhibits infections by strains of cucumber mosaic virus; inhibition is greatest when the enzyme is present in the inoculum, and when applied to inoculated leaves its effect decreases rapidly with increasing time after inoculation.
Infection by and the multiplication of strains of cucumber mosaic virus in tobacco are only slightly affected by thiouracil and greatly by azaguanine, whereas strains of tobacco mosaic virus are inhibited much more by thiouracil than by azaguanine. Like tobacco mosaic virus, cucumber mosaic virus multiplies more when inoculated leaves are floated in nutrient solutions than in water, but unlike tobacco mosaic virus, its multiplication is not inhibited by thiouracil more in nutrient solutions than in water.     

SAP-TRANSMISSIBLE MOSAIC DISEASES OF SOLANACEOUS CROPS IN TRINIDAD

Four sap-transmissible viruses were isolated from cultivated Solanaceae in Trinidad: (1) tobacco mosaic virus, from tobacco, tomato and sweet pepper; (2) cucumber mosaic virus, from tobacco and petunia; (3) 'pepper vein-banding virus', probably related to pepper mosaic viruses in Puerto Rico and Brazil, from peppers and tobacco; (4) 'egg-plant mosaic virus', possibly related to the tobacco ring-spot virus, from egg-plant and tomato. Pepper vein-banding virus causes leaf-crinkling and vein-banding in Physalis floridana , petunia, various Nicotiana spp. and most peppers; the Large Bell Hot pepper is killed; tomato and egg-plant are immune. Egg-plant mosaic virus produces mosaic, ring-spotting, or both, on different solanaceous species. It also gives local and systemic ring-spotting on Chenopodium hybridum and necrotic local lesions on the primary leaves of cowpea (var. Black-eye); cucumber is a symptomless carrier. Only cucumber mosaic virus was found naturally infecting non-solanaceous hosts, cucumber and certain common wild plants.
The thermal inactivation point of pepper vein-banding virus is 62° C, its dilution end-point 2×10^-5 and its longevity in vitro 6 day s at 23–30° C.; corresponding values for egg-plant mosaic virus are 78° C., 10^-6 and over 3 weeks. Aphisgossypii transmits cucumber mosaic and pepper vein-banding, but not egg-plant mosaic, of which Epitrix sp. is an occasional vector. Tobacco mosaic, as elsewhere, probably has no regular insect vectors in Trinidad.     

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.     

ANEMONE MOSAIC–A VIRUS DISEASE

The name anemone mosaic is proposed for a previously unrecorded virus disease of Anemone coronaria L.; infected plants have mottled leaves, and broken and distorted flowers. This virus can cause winter browning, and can contribute to crinkle in anemones.
The virus infected forty-seven out of ninety plant species tested; it was transmitted by mechanical inoculation, and by four of the six aphid species tested. Most aphids ceased to be infective within 30 min. when continuing to feed after leaving an infected plant.
Properties in vitro varied according to conditions of the tests; the thermal inactivation point was always below 62°C., the dilution end-point did not exceed 1/2500, and the virus inactivated at 18°C., the fewer than 72 hr.
Intracellular inclusion bodies were produced in all hosts examined.
Anemone mosaic virus is very similar to viruses placed in the turnip virus 1 group of Hoggan & Johnson, and is serologically related to cabbage black ringspot virus, although AMV infection did not protect plants against infection with cabbage black ring-spot virus.
Weeds naturally infected with AMV were found in anemone plantations, and this virus was detected, together with cucumber mosaic and tobacco necrosis viruses, in corms imported into this country.     

RASPBERRY YELLOW DWARF, A SOIL-BORNE VIRUS

An apparently undescribed virus, provisionally named raspberry yellow dwarf virus (RYDV), was isolated from naturally infected raspberry, strawberry, blackberry and several weed species by mechanical inoculation of sap to Chenopodium amaranticolor. The severe disease it caused in Malling Exploit raspberry usually occurred patchily in otherwise normal plantations: these patches increased in size from year to year. RYDV was differentiated from raspberry ringspot and tomato black ring viruses by the symptoms produced in C. amaranticolor , tobacco and Petunia hybrida. RYDV lost infectivity when sap was heated for 10 min. at 61° C., diluted 10^-5or kept for 15 days at 18° C. RYDV was precipitated without inactivation by acetone and by ammonium sulphate.
Isolates of RYDV from different plants and localities, and of different virulence, were identified by plant-protection and serological tests. Such tests gave no evidence that RYDV was related to raspberry ringspot, tobacco ringspot, tomato black ring or cucumber mosaic viruses.
Raspberry and sugar-beet plants became systemically infected with RYDV when grown under glass in soil from a field where the disease had occurred in raspberry plants, and where the virus persisted in the soil for 3 years after the raspberry plants were removed. RYDV seems to be widely disseminated in England but recently introduced and rare in eastern Scotland.
Like raspberry ringspot and tomato black ring viruses, RYDV causes symptoms of the ringspot type in tobacco, has a wide natural and experimental host range, is soil-borne and of local importance. Such features seem characteristic of ringspot viruses as a group.     

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.     

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.     

PROPERTIES AND HOST RANGE OF TURNIP CRINKLE, ROSETTE AND YELLOW MOSAIC VIRUSES

Three isolates of turnip yellow mosaic virus and two other flea-beetle transmitted viruses, turnip crinkle and turnip rosette, have many similar properties: thermal inactivation end-point between 80 and 90° C.; dilution end-point greater than 10^-4; longevity in vitro at about 20° C. at least 30 days. All were transmitted by mechanical inoculation to a wide range of cruciferous host plants, including many weeds. Turnip yellow mosaic virus infected only Reseda odorata outside the Cruciferae , whereas rosette virus infected a few and crinkle virus many non-cruciferous hosts.     

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.     

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.     

SOME EFFECTS OF HOST NUTRITION ON THE SUSCEPTIBILITY OF PLANTS TO INFECTION BY CERTAIN VIRUSES

Fertilizer treatments that greatly influenced the growth of tobacco and potato plants in pots had little effect on the number that became infected with potato virus Y when the plants were colonized by equal numbers of infective aphids, though the number was slightly decreased by nitrogen and increased by phosphorus.
The number of local lesions produced on leaves of tobacco and Nicotiana glutinosa by tomato aucuba mosaic and tobacco mosaic viruses was increased by additions of both nitrogen and phosphorus, provided that these also increased growth. The predominant effect of both nutrients in increasing susceptibility was indirect by increasing plant size, but over certain critical ranges both elements also increased the numbers of lesions produced per unit leaf area. Conditions of maximum susceptibility approximated closely to those producing optimal growth, and susceptibility, whether measured by lesions per half-leaf or per unit area, was decreased by a deficiency or excess of either element. Sometimes the addition of nitrogen reduced susceptibility when still increasing plant growth.     

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.     

The Systemic Infection by Tobacco Mosaic Virus of Tobacco Plants Coutaining the N Gene at Temperatures Below 28°C

Tobacco plants containing the N-gene are occasionally systemically infected with tobacco mosaic virus (TMV) at tempreratures below 28°C, but contain low concentrations of virus: they often fail to set seed, and can outlive healthy control plants. Infection is thus similar to that induced when N-gene tobacco plants are grafted onto systemically infected tobacco lacking the N-gene. Shoots from systemically infected N-gene plants can induce systemic infection in other graft-inoculated N-gene plants. Stem sections of N-gene tobacco plants act as good conduits for TMV between plants lacking the N-gene, and girdling experiments suggest that virus movement probably occurs in the phloern.     

Tobamovirus coat protein CPCg induces an HR-like response in sensitive tobacco plants

When inoculated into sensitive tobacco Xanthi-nn plants, the crucifer and garlic-infecting Tobacco mosaic virus (TMV-Cg) induces local necrotic lesions that resemble those seen in the hypersensitive response (HR) of resistant tobacco plants. However, unlike these, tobacco Xanthi-nn plants do not become resistant to infection and the virus spreads systemically causing a severe disease characterized by necrotic lesions throughout the plant. To identify the viral protein that elicits this necrotic response, we used a set of hybrid viruses constructed by combination of TMV-Cg and the tobacco mosaic virus strain U1 (TMV-U1). In this study we present evidence that the coat protein of TMV-Cg (CPCg) is the elicitor of the necrotic response in tobacco Xanthi-nn plants. Local and systemic necrotic lesions induced by TMV-Cg and by the hybrid U1-CPCg -that carries CPCg in a TMV-U1 context- are characterized by cell death and by the presence of autoflorescent phenolic compounds and H2O2, just like the HR lesions. In addition, defense-related genes and detoxifying genes are induced in tobacco Xanthi-nn plants after TMV-Cg and U1-CPCg inoculation. We postulate that in our system, CPCg is recognized by sensitive tobacco plants that mount an incomplete defense response. We call this an HR-like since it is not enough to induce plant resistance.     

THE INFECTION OF PLANTS BY VIRUSES THROUGH ROOTS

Roots of young tomato plants became infected when inoculated with tomato bushy stunt, tobacco mosaic, and potato X viruses. Root infections also occurred when these viruses were added to soil or culture solutions in which plants were growing.
The viruses were sometimes localized around their initial entry points in roots; sometimes they invaded the root system but not the shoots, and sometimes they produced full systemic infection of roots and shoots. In some experiments, but not all, systemic infections were more frequent when the upper tap root or superficial roots were inoculated than when fibrous roots were inoculated.
In both tomato and potato, virus X spread from diseased to healthy plants sharing the same culture solution, if their roots were in contact, but not otherwise. Infection of the roots of potato plants by inoculation, produced only one plant with virus-infected haulms, although several had infected tubers.     

STUDIES ON THE HOST RANGE, PROPERTIES AND MODE OF TRANSMISSION OF BEET RINGSPOT VIRUS

An apparently undescribed mechanically transmissible virus has been named beet ringspot virus (BRV). It occurs naturally in Scotland in sugar-beet, turnip, swede, potato and many kinds of weed plants. BRV is readily distinguished from raspberry ringspot virus by the symptoms produced in Chenopodium amaranticolor , French bean, tobacco and Petunia hybrida plants. BRV lost infectivity when heated for 10 min. at 63°C. but not at 60°C.: at 20°C. its longevity in vitro was about 3 weeks. BRV was precipitated by ammonium sulphate, ethanol and acetone.
Protection experiments with tobacco plants, and serological tests, gave no evidence that BRV was related to tobacco ringspot, raspberry ringspot, potato bouquet or tobacco rattle viruses, but showed that viruses isolated from different host plants and from different localities were strains of BRV.
BRV is soil-borne: in glasshouse experiments sugar-beet, beetroot, potato, turnip, swede, French bean, Fragaria vesca , oat and wheat plants often became systemically infected when grown in soil from the site of a disease outbreak, but the virus was restricted to the roots of many infected plants. When sugar-beet seedlings were grown in virus-containing soil, BRV was first detected in their roots, where its concentration increased, before progressively increasing amounts of virus were found in the shoots.
Soils from five localities were found to contain BRV. BRV has been found only where the soil is light in texture, and often in fields where raspberry ringspot virus occurs.     

THE PROPERTIES OF TOMATO ASPERMY VIRUS AND ITS RELATIONSHIP WITH CUCUMBER MOSAIC VIRUS*

Chrysanthemum plants infected with tomato aspermy virus (TAV) produce severely distorted and discoloured flowers but show only slight leaf mottle.
TAV infected twenty-five of forty-five species (belonging to seventeen genera) tested and was transmitted by the aphid species Aulacorthum solarti, Macrosiphoniella sanborni and Myzus persicae .
Sap from infected tobacco leaves lost infectivity when diluted more than 1 in 10,000, when heated for 10 min. at above 65°C. and when stored for more than 42 hr. at 16–18°C.
Partial protection was obtained between TAV and two strains of cucumber mosaic virus. Evidence was obtained that this was true protection between related viruses and serological tests confirmed the view that TAV is a strain of cucumber mosaic virus. Evidence was obtained that this was true protection between related viruses and serological tests confirmed the view that TAV is a strain of cucumber mosaic virus.