Properties of some defective strains of tobacco mosaic virus and their behaviour as affected by inhibitors during storage in sap

From the type strain of tobacco mosaic virus, defective strains were isolated that produced chlorotic or ringspot type symptoms in tobacco and were difficult to transmit without carborundum in the inoculum. Their concentration was less than 0–1 μg/ml of sap instead of the usual 2 mg/ml with the type strain. Phenol extracts of infected leaves were a little more infective than extracts in buffer, whereas phenol extracts of leaves infected with type strain were very much less infective than extracts in buffer. Electron microscopy of infective sap rarely showed any virus particles, but preparations concentrated by ultracentrifugation contained virus particles, many of which were broken or seemed inadequately assembled. Changing the ambient temperature at which infected plants were kept from 20 to 35°C did not increase the amount or improve the appearance of the virus. Some of the strains were inactivated during heating for 10 min between 70 and 80 °C. Undiluted sap lost its infectivity in 3 days at 20 °C, as did the type strain when diluted to 0–1 μg/ml in sap from healthy leaves. This is because substances that inhibit infection were produced by microbes in the sap. The ability of sap from healthy leaves to inhibit infection increased by more than twenty-five times when left 3 days at 20 °C. Infectivity of appropriate mixtures of type strain and aged sap was restored by diluting them in buffer. Sodium azide at 0·02% in sap prevented formation of the inhibitor. The infectivity of the defective strains increased when inoculated together with the type strain.     

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 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.     

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.     

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.     

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 DISTRIBUTION OF VIRUSES IN LEAVES AND THEIR INACTIVATION BY ULTRA-VIOLET IRRADIATION

The infectivity of sap expressed from the lower epidermis stripped from leaves systemically infected with potato virus Y , henbane mosaic virus or tobacco mosaic virus was compared with that of sap from the underlying mesophyll. Results suggested that the concentration of virus in each of the two tissues was about the same.
Ultra-violet irradiation of leaves infected with potato virus Y or henbane mosaic virus greatly reduced the infectivity of sap expressed from subepidermal tissues.     

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.     

THE DISTRIBUTION OF VIRUSES IN DIFFERENT LEAF TISSUES AND ITS INFLUENCE ON VIRUS TRANSMISSION BY APHIDS

Exposing both surfaces of leaves systemically infected with cabbage black ring spot virus (CBRSV) or henbane mosaic virus to ultra-violet radiation decreases the infectivity of expressed sap to about one-fifth. As irradiation probably inactivates virus mainly in the epidermis, which occupies about one-quarter the volume of the leaves, these viruses seem to occur at much higher concentrations in sap from the epidermis than in sap from other cells. By contrast, tobacco mosaic virus seems not to occur predominantly in the epidermis.
CBRSV and henbane mosaic virus are normally transmitted most frequently by previously fasted aphids that feed for only short periods on infected leaves, but aphids treated like this transmit rarely from leaves that have been exposed to ultraviolet radiation. Irradiation has relatively little effect on the proportion of aphids that transmit after long infection feedings. Fasting seems to increase transmission by increasing the probability that aphids will imbibe sap from the epidermis of leaves they newly colonize. With longer periods on infected leaves, the ability of fasted aphids to transmit probably decreases because they then feed from deeper cells and their stylets contain sap with less virus. Only virus contained in the stylets seems to be transmitted, not virus taken into the stomach. About half the transmissions of henbane mosaic virus by aphids that have colonized tobacco leaves for hours may be caused by insects that temporarily cease feeding on the phloem and newly penetrate the epidermis.
Irradiating infected leaves affected the transmission of sugar-beet mosaic virus in the same way as that of henbane mosaic virus, but had little effect on the transmission of beet yellows virus, whose vectors become more likely to transmit the longer they feed on infected plants.     

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.     

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.     

CERTAIN ENZYMATIC ACTIVITIES OF NORMAL AND MOSAIC INFECTED TOBACCO PLANTS

The lower leaves of tobacco plants were inoculated with leaf mosaic virus and the activities of oxygenase, peroxidase, catalase, and invertase were followed in leaves of comparable age at intervals of 2 or 3 days over a period of 21 days. The inoculated leaves exhibited a great decrease relative to normal tissue in the activity of oxygenase and peroxidase on the 6th day. Younger leaves showed this minimum at a progressively later date. A great decrease in the activities of these enzymes was attained by the 14th to the 18th day. This maximum was followed by a decrease. Catalase exhibited an increased activity which reached a maximum at about the 8th day. A second maximum was observed on the 16th to the 18th day. Invertase reached a minimum, relative to normal plants, on about the 8th day. A second minimum was approached on the 16th to the 18th day. These data show that profound disturbances in the physiology of infected plants occur many days before the leaf juice attains an infectious concentration of virus. The observed activities could not be due therefore to metabolic activities of the virus particles themselves. Since infectivity is attained only after a period of profound physiological disturbance, it seems possible that the virus protein develops as a product of abnormal metabolism.     

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.     

STREAK—A VIRUS DISEASE OF TOMATOES

A comparison of streak disease of tomatoes, derived from commercial glasshouses, and experimental streak produced by combined inoculation of the viruses of potato mosaic and tobacco mosaic, is given in detail.
The characters employed in comparison are the host range of each virus and its resistance to various temperatures, to different concentrations of alcohol, and to ageing in vitro .
Glasshouse streak and tobacco mosaic show an equal resistance to alcohol, heat and ageing in vitro , and have, in addition, an identical host range. Treatment for 1 hour with 90 per cent, alcohol and for 10 minutes at 85⁰ C. did not destroy the infectivity of either of these viruses.
Glasshouse streak is shown not to contain the virus of potato mosaic, but is of itself able to produce necrosis in tomatoes without the participation of potato mosaic. The factors underlying this have not been determined.
It is concluded that tobacco mosaic and the mosaic of glasshouse streak are probably identical, and that much of the streak occurring in glasshouses is due to a single virus, and not a mixed infection of this with potato mosaic.     

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.     

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.     

广东省烟草花叶病病原病毒的鉴定

烟草花叶病是广东省产烟区的主要病害之一。我们在南雄等八个县进行调查,1983年至1984年的一般发病率为2～20％。根据血清学反应、病毒粒体形态、鉴别寄主反应及寄主范围、媒介昆虫种类、物理性质、交互保护反应等各项检验结果,鉴定广东省烟草花叶病病原是:三个黄瓜花叶病毒可能株系(普通株系CMV-C,烟草坏死株系CMV-TN,烟草黄色坏死株系(CMV-TYN),烟草花叶病毒(TMV),马铃薯病毒Y(PVY),烟草脉带花叶病毒(TVBMV)和烟草褪绿斑驳病毒(TCMV)(暂定)。     

Use of protein A to improve sensitisation of latex particles with antibodies to plant viruses

Protein A-coated latex (PAL) was compared with uncoated latex (L) for sensitisation with antibodies to five plant viruses: apple mosaic virus (ApMV), arabis mosaic virus (AMV), plum pox virus (PPV), potato virus Y ordinary strain (PVY°) and prunus necrotic ringspot virus (NRS V). A range of globulin concentrations was used with each antiserum and detection end points determined in serial dilutions of infective sap. When sensitised with antibodies to ApMV, PAL detected ApMV readily, whereas L did not. When sensitized with antibodies to PVY° and AMV, PAL gave higher detection end points than L. However, PAL gave little increase in sensitivity with the antisera to PPV and NRSV. Non-specific aggregation of latex, which sometimes occurred in very dilute sap with PAL, could be dispersed by adding 0.02% Tween-20 to the extraction buffer. Globulins of PVY° and AMV could be used at higher dilutions with PAL than with L, giving a saving in antiserum. Both types of latex sensitised with PVY° antibody globulins readily detected the tobacco veinal necrosis and C strains of this virus.     

Transmission characteristics and some other properties of bean yellow vein-banding virus, and its association with pea enation

Bean yellow vein-banding virus (BYVBV) has been found occasionally in mixed infection with pea enation mosaic virus (PEMV) in spring-sown field beans (Vicia faba minor) in southern England. Glasshouse tests confirmed that, like PEMV, BYVBV is transmissible by manual inoculation and by aphids in the persistent manner. However, BYVBV can be transmitted by aphids only from plants that are also infected with a helper virus, usually PEMV. Thus after separation from PEMV by passage through Phaseolus vulgaris it was no longer aphid-transmissible. It became aphid-transmissible again only after re-mixing in plants with PEMV or with a substitute helper, bean leaf roll virus (BLRV). It was not transmitted by aphids that fed sequentially on plants singly infected with PEMV and BYVBV. Thus the interaction between BYVBV and PEMV (or BLRV) that enables BYVBV to be transmitted by aphids seems to occur only in doubly infected plants. However, it was not transmitted by aphids from plants doubly infected with BYVBV and broad bean wilt virus (BBWV). BYVBV and PEMV were transmitted more readily by Acyrthosiphon pisum than by Myzus persicae; neither virus was transmitted by Aphis fabae. Phenol extracts of BYVBV-infected leaves were more infective than phosphate buffer or bentonite-clarified extracts and were sometimes infective when diluted to 1/1000. The infectivity of BYVBV in phosphate buffer extracts of leaves singly infected with BYVBV, unlike that in extracts of leaves doubly infected with BYVBV and PEMV (or BLRV), was destroyed by treatment with organic solvents. BYVBV infected 11 of 28 plant species that were inoculated with phenol extracts; seven of the infected species were legumes. No transmission of BYVBV was detected through seed harvested from infected field bean plants. Isometric particles c. 30 nm in diameter were seen in extracts of plants doubly infected with BYVBV and PEMV but not in extracts of plants infected with BYVBV alone. Leaves of plants infected with BYVBV, alone or with PEMV, contained membrane-bound structures c. 50–90 nm in diameter associated with the tonoplast in cell vacuoles. These structures were not found in healthy leaves. BYVBV has several properties in common with other known aphid-borne viruses that are helper-dependent and transmitted in a persistent manner. Possibly, as suggested for some of them, aphid transmission of BYVBV depends on the coating of its nucleic acid with helper virus coat protein.     

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.