Electron microscopical proof of the presence of sugar beet yellows virus particles in the roots of sugar beet

The presence of beet yellows virus (BYV) particles was electron microscopically proved in the roots of sugar beet. Specimens for the electron microscopical examination of root sap were prepared by differential centrifugation. It was proved that, contrary to expectations, examinations in spring showed most virus particles in the basal part of the root. At the same time it was found by experiment that the diagnostical BYV antiserum, for which the antigen was prepared from sugar beet leaves, did not react with a purificate of BYV containing virus particles.     

THE TRANSMISSION BY MITES, HOST-RANGE AND PROPERTIES OF RYEGRASS MOSAIC VIRUS

A virus that causes chlorotic streaks on ryegrass leaves was transmitted by the eriophyid mite Abacarus hystrix (Nalepa). Virus-free mites acquired the virus in 2 hr. feeding on infected ryegrass and the proportion that became infective increased with increased feeding time up to 12 hr.; vectors lost infectivity within 24 hr. of leaving the infected leaves. All instars of A. hystrix transmitted the virus.
The virus was transmitted by manual inoculation of sap to other species of Gramineae, including oats, rice, cocksfoot and meadow fescue, but none of these hosts seemed to contain as much virus as ryegrass; their saps did not precipitate specifically with antiserum prepared against the virus in ryegrass, whereas sap from infected ryegrass precipitated up to a dilution of 1/32. Infective sap of S22 Italian ryegrass contained flexuous rod-shaped particles; the dilution end-point of the virus was about 1 in 1000; the virus was inactivated when held for 10 min. at 60°C. and most of its infectivity was lost after 24 hr. at room temperature.     

Comparison of the contents of sucrose and amino acids in the leaves, phloem sap and taproots of high and low sugar-producing hybrids of sugar beet (Beta vulgaris L.)

The contents of sucrose and amino acids in the leaves, phloemsap and taproots have been analysed in three experimental hybridsof sugar beet and compared with earlier analysed leaf and phloemsap contents in spinach and barley. The three hybrids accumulatedsucrose and amino acids to various extents in the mature rootsas well as in the young taproots (9–12 weeks). The differencesin the sucrose-to-amino acid ratios in the taproots were reflectedin the corresponding ratios in the phloem sap. The leaf contentsof sucrose and amino acids in the three hybrids were found tobe very similar to each other and also to those in spinach andbarley. In contrast, the phloem concentration of sucrose (1.3M) was much higher, and that of amino acids much lower thanin spinach and barley. In the taproots, the overall concentrationof sucrose was about half that in the phloem sap. From thesefindings it is con cluded that the decisive factor in the highsucrose accumulation in sugar beet roots is the very efficientprocess of phloem loading in the leaves. The patterns of theamino acids in the phloem sap and in the taproots resembledthose in the leaves, indicating that there is no special transportform for a-amino nitrogen from the leaves to the roots, butall amino acids which are present in the cytosol are translocated. Key words: Amino acids, Beta vulgaris L., phloem sap, sucrose, tap roots, transport     

Influence of phloem transport, N-fertilization and ion accumulation on sucrose storage in the taproots of fodder beet and sugar beet

To investigate the factors governing the accumulation of sucroseand amino acids in the taproots of sugar beet, their contentswere measured in the leaves, phloem sap and the taproots ofsugar beet, fodder beet and a hybrid between both, grown oneither 3.0 or 0.5 mM nitrate. In the taproots the contents ofmalate, citrate and inorganic ions were also determined. Forthe high sucrose accumulation in sugar beet as compared to theother varieties three factors were found. (a) In sugar beet,less amino acids and more sucrose are taken up into the phloemthan in fodder beet. (b) In sugar beet, the sucrose and aminoacid syntheses are less sensitive to the nitrate concentrationsthat are required for optimal plant growth than in other varieties.In fodder beet, upon raising the nitrate concentration from0.5 mM to 3 mM, the synthesis and storage of sucrose is decreasedand that of amino acids increased. The corresponding valuesin sugar beet (0.5 mM) are similar to those in fodder beet andare not much affected by an increase of nitrate. (c) The sucroseaccumulation is limited by the accumulation of inorganic ionsin the taproots. The sucrose content in the taproots is negativelycorrelated to the total ion content. Whereas sucrose representstwo-third of all solutes in the taproots of sugar beet, it amountsto only one-third of the solutes in fodder beet taproots. Key words: Amino acids, Beta vulgans L, phloem sap, potassium, sucrose storage, sugar beet, taproots, transport     

SOME PROPERTIES OF BROAD-BEAN MOTTLE VIRUS

A severe disease affecting many plants in a crop of broad beans was found to be caused by a previously undescribed virus, provisionally named broad-bean mottle virus. The distribution of diseased plants suggested spread by a vector, but none of the six insects tested transmitted it. The virus was transmitted to several species of leguminous plants by mechanical inoculation of sap; infectivity for some hosts seemed to be increased by propagation in these hosts.
The virus has an unusual combination of properties. Its thermal inactivation point is about 95°C., whereas sap becomes non-infective within 3 weeks at room temperature. The infection end-point of broad-bean sap is 1/1000, only a little higher than the precipitation titre with specific antiserum. Precipitation with antiserum occurs over a smaller range of antigen/antibody ratios than with other viruses previously studied, possibly because of its greater solubility; it is not precipitated with (NH₄)₂SO₄ until the salt concentration exceeds 75% saturation.
A specific nucleoprotein, containing nucleic acid of the ribose type, can be isolated from infective broad-bean sap in yields up to 2 g./l. Purified preparations, made by salt precipitation and ultracentrifugation, contain uniform spherical particles approximately 17 mμ in diameter. It is suggested that much of this nucleoprotein is non-infective, but may otherwise resemble infective particles.     

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.     

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.     

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.     

Effects of iron deficiency on the composition of the leaf apoplastic fluid and xylem sap in sugar beet. Implications for iron and carbon transport

The effects of iron deficiency on the composition of the xylem sap and leaf apoplastic fluid have been characterized in sugar beet (Beta vulgaris Monohil hybrid). pH was estimated from direct measurements in apoplastic fluid and xylem sap obtained by centrifugation and by fluorescence of leaves incubated with 5-carboxyfluorescein and fluorescein isothiocyanate-dextran. Iron deficiency caused a slight decrease in the pH of the leaf apoplast (from 6.3 down to 5.9) and xylem sap (from 6.0 down to 5.7) of sugar beet. Major organic acids found in leaf apoplastic fluid and xylem sap were malate and citrate. Total organic acid concentration in control plants was 4.3 mM in apoplastic fluid and 9.4 mM in xylem sap and increased to 12.2 and 50.4 mM, respectively, in iron-deficient plants. Inorganic cation and anion concentrations also changed with iron deficiency both in apoplastic fluid and xylem sap. Iron decreased with iron deficiency from 5.5 to 2.5 microM in apoplastic fluid and xylem sap. Major predicted iron species in both compartments were [FeCitOH](-1) in the controls and [FeCit(2)](-3) in the iron-deficient plants. Data suggest the existence of an influx of organic acids from the roots to the leaves via xylem, probably associated to an anaplerotic carbon dioxide fixation by roots.     

Properties of cocksfoot streak and cocksfoot cryptic, two viruses infecting cocksfoot (Dactylis glomerate) in Scotland

A Scottish isolate of cocksfoot streak virus (CSV-S) was found to have flexuous filamentous particles which, in sap of infected cocksfoot plants, had a modal length of 712 nm. It was transmitted from infected to healthy cocksfoot plants in a non-persistent manner by Myzus persicae and by mechanical inoculation of infective sap extracts containing an anti-oxidant. Apart from cocksfoot, mechanical inoculation of infective sap succeeded in infecting only four of 22 plant species tested. The infectivity of sap extracts containing 0.2% thioglycerol was lost after heating for 10 min at 55^oC but not 50^oC, storage at room temperature for 48 but not 24 hours, and after diluting 10-2 to 10-3. Highly purified preparations of CSV-S particles sedimented as a single component with a sedimentation coefficient of 139S and had a buoyant density in rubidium bromide of 1.31 g/cm3. Virus particles were composed of one protein and one ssRNA species with estimated Mr of 31 000 and 3.2 times 106 respectively. In ELISA, an antiserum prepared to CSV-S detected the virus in all aerial parts of infected cocksfoot plants and, when present in the ratio of 1 infected leaf: 1000 healthy leaves. Both CSV-S-infected and -uninfected cocksfoot also contained a previously undescribed virus with isometric particles c. 30 nm in diameter. This virus, named cocksfoot cryptic virus (CCV), was seed-borne in two cvs of cocksfoot tested and its particles contained two dsRNA species of estimated Mt of 1.14 times 106 and 1.27 times 106. Despite the fact that particles of CSV-S were largely free from CCV particles following exclusion chromatography on agarose beads prior to immunisation, immunoelectron microscopy (IEM) showed that the antiserum prepared to CSV-S also contained some antibodies to CCV. Evidence from IEM suggested a possible distant serological relationship of CCV to ryegrass and beet (BCV 1 or BCV 2, or both) cryptoviruses, all members of sub-group A of cryptoviruses.     

Nicotiana velutina mosaic virus: purification, properties and affinities with other rod-shaped viruses

Nicotiana velutina mosaic virus (NVMV), found in Australia, was transmitted by inoculation of sap to twenty species in the Solanaceae and Chenopodiaceae, and to Gomphrena globosa; its host range closely resembles that of potato mop-top virus (PMTV). Infectivity was abolished when sap was kept at room temperature between 1 and 4 days, or when heated for 10 min between 60 and 70 °C. NVMV was frequently transmitted through the seed of four Nicotiana spp. NVMV and PMTV were purified by a method that involved redissolving virus particles sedimented by low speed centrifugation of leaf extracts, followed by sedimentation through sucrose cushions. NVMV preparations contain rod-shaped particles about 18 nm wide and with a large range of lengths, the commonest being 125–150 nm. The particles have a helical structure with a pitch of 2–9 nm, break easily, and contain a single protein of apparent mol. wt. 21|400, slightly larger than that of PMTV (19 800). In serological tests assessed by electron microscopy, no relationship was detected between NVMV and PMTV, or barley stripe mosaic, beet necrotic yellow vein, soil-borne wheat mosaic, tobacco mosaic or tobacco rattle viruses. However, antiserum to soil-borne wheat mosaic virus reacted quite strongly with PMTV and weakly with tobacco mosaic virus. NVMV is considered to be a distinct member of the tobamovirus group; its frequent transmission through seed may be an adaptation to the arid environment where it was found. Its cryptogram is */*:*/*:E/E:S/*.     

A VIRUS DISEASE OF RED CURRANT (RIBES RUBRUM L.)

A virus causing ringspot patterns and vein clearing of red currant leaves is reported. It is inactivated at 66–68°C., tolerates a dilution of 1:2000 but not 1:5000, loses infectivity after 7–9 days in vitro and has been transmitted only by sap inoculation.
The virus infects plants in a wide range of families, resulting in chlorotic and necrotic symptoms. On Nicotiana tabacum it causes local and systemic symptoms of the ringspot type. Its host range and physical properties differ from other viruses causing ringspot symptoms, and the name red currant ringspot virus is therefore suggested.     

THE INFLUENCE OF TEMPERATURE AND DAYLENGTH ON SPOTTED WILT VIRUS DISEASE OF TOMATO

The responses of young tomato plants to infection with tomato spotted wilt virus were studied at constant temperatures. The plants were grown in nutrient solutions and given three daylength treatments.
The length of the incubation period was positively correlated with temperature and a regression equation for this relationship is given. The calculated temperature at which this period becomes infinite is 6.7°C. The temperature coefficient ( Q ₁₀) varied from 1.7 to 2.9.
Dry-weight determinations were made of stems, leaves and roots. In healthy plants the leaf/stem ratio generally fell with rise of temperature and the shoot/root ratio rose. Infection changed these ratios, which is interpreted as indicating that it interfered with supplies of carbohydrate to the growing regions.
Systemic symptoms are described for plants raised at 11.4°, 18.3°, 23.9°, and 29.4°C.
Changes in daylength did not affect the systemic symptoms or incubation period. With a 9 hr. day at 29.4°C., the virus content of sap was significantly lower than with 12 or 15 hr., and with a 12 hr. day was significantly higher than with a 15 hr. day.     

Natural infection of sugar beet with tomato bushy stunt virus

A virus transmissible toChenopodium quinoa was isolated from leaves of sugar beet showing large chlorotic ring spots and line pattern. The virus was serologically unrelated to tobacco necrosis virus and tomato black ring virus or to its beet ringspot strain either. A positive result was obtained with antiserum against tomato bushy stunt virus. Reactions of herbaceous indicators and properties of the virus in crude sap were in accordance with the serological diagnosis. A survey of natural hosts of tomato bushy stunt virus demonstrated recently by the authors is given.     

Responses of sugar beet (Beta vulgaris L.) to drought and nutrient deficiency stress

The responses of two sugar beet genotypes, 24367 (putative droughttolerant) and N6 (putative drought intolerant), to drought and nutrientdeficiency stress were investigated in an attempt to identify reliable andsensitive indicators of stress tolerance. In glasshouse-grown plants of bothgenotypes, relative water content (RWC) of the leaves decreased and leaftemperature increased in response to drought stress. Genotype differences inresponse to drought included leaf RWC, glycine betaine accumulation, alterationof shoot/root ratio and production of fibrous roots. Thus, in comparison to N6,genotype 24367 lost less water from leaves, produced more fibrous roots,produced more glycine betaine in shoots and tap roots and had a much reducedshoot/root ratio in response to withholding water for up to 215 h.The hydraulic conductance and sap flow of sugar beet seedlings grown innutrientculture decreased when subjected to nitrogen deficiency stress. Under nitrogensufficient conditions sap flow was greater in 24367 than in N6. The resultsindicate that genotype 24367 is more tolerant to stresses induced by water andnitrogen deficiency and that increased fibrous root development may be a majorfactor in increasing sap flow via a concomitant enhancement of aquaporinactivity.     

Use of DNA from dry leaves for PCR and RAPD analysis

Fresh or frozen tissue is usually used as a source of DNA for PCR and RAPD analysis. We have found that leaves can be allowed to dry at room temperature before extraction of DNA. Heating the leaves or microwave drying resulted in poor recovery of DNA. Storage of fresh leaves in paper envelopes in the laboratory was the most successful approach. This allowed the tissue to dry out over a period of several days and DNA could be extracted at any time, providing a convenient method for the collection and analysis of field material. DNA from leaves stored for four months at room temperature was suitable for PCR analysis.     

Purification and conservation of infective sugar beet mosaic virus

Sugar beet mosaic virus (SBMV) was precipitated by polyethyleneglycol (PEG) 6000 from the cell sap of infected sugar beet leaves. After centrifugation and addition of dextrane T 10 the virus was lyophilized. Its infectious activity was demonstrated by mechanical transmission toChenopodium quinoa Willd. and to sugar beet. Stability of infectious activity of the lyophilized virus was verified.     

Properties of cocksfoot streak and cocksfoot cryptic, two viruses infecting cocksfoot (Dactylis glomerata) in Scotland

A Scottish isolate of cocksfoot streak virus (CSV-S) was found to have flexuous filamentous particles which, in sap of infected cocksfoot plants, had a modal length of 712 nm. It was transmitted from infected to healthy cocksfoot plants in a non-persistent manner by Myzus persicae and by mechanical inoculation of infective sap extracts containing an anti-oxidant. Apart from cocksfoot, mechanical inoculation of infective sap succeeded in infecting only four of 22 plant species tested. The infectivity of sap extracts containing 0.2% thioglycerol was lost after heating for 10 min at 55^oC but not 50^oC, storage at room temperature for 48 but not 24 hours, and after diluting 10^-2to 10^-3. Highly purified preparations of CSV-S particles sedimented as a single component with a sedimentation coefficient of 139S and had a buoyant density in rubidium bromide of 1.31 g/cm³. Virus particles were composed of one protein and one ssRNA species with estimated M_r of 31 000 and 3.2 times 10⁶respectively. In ELISA, an antiserum prepared to CSV-S detected the virus in all aerial parts of infected cocksfoot plants and, when present in the ratio of 1 infected leaf: 1000 healthy leaves. Both CSV-S-infected and -uninfected cocksfoot also contained a previously undescribed virus with isometric particles c. 30 nm in diameter. This virus, named cocksfoot cryptic virus (CCV), was seed-borne in two cvs of cocksfoot tested and its particles contained two dsRNA species of estimated M_r of 1.14 times 10⁶and 1.27 times 10⁶. Despite the fact that particles of CSV-S were largely free from CCV particles following exclusion chromatography on agarose beads prior to immunisation, immunoelectron microscopy (IEM) showed that the antiserum prepared to CSV-S also contained some antibodies to CCV. Evidence from IEM suggested a possible distant serological relationship of CCV to ryegrass and beet (BCV 1 or BCV 2, or both) cryptoviruses, all members of sub-group A of crypto viruses.     

Thermographic analysis of leaf water and energy information of Japanese spindle and glossy privet trees in low temperature environment

Thermography has been used in many fields to perform non-invasive temperature measurements of natural objects. In this paper, thermography was used to determine the temperature of leaves, stems and branch kerfs of Japanese spindle (Euonymus japonicus Thunb.) and glossy privet (Ligustrum lucidum Ait.) in the city of Jinan in China during winter. The temperatures of the leaves, stems and branch kerfs were monitored as the temperature decreased after the sample was subjected to hand heating or after the branch was cut. Differences in the specific heats and the latent heats of the leaves, branches and stems with different water contents and transpiration capacities were confirmed. The significant temperature difference obtained after hand heating between different leaf sections with varied water contents made it easy to obtain the thermal images, which were clear and exhibited reduced systematic errors. After hand heating, a significantly higher temperature was found at the major vein system of both Japanese spindle and glossy privet. This increased temperature difference made it possible to detect the water and the thermal state of these leaves. Therefore, it was possible to detect scorched area of the leaves, the twig dieback and the sap warming phenomenon in the leaves using thermography. In addition, the leaf bending phenomenon observed in Japanese spindle leaves during the deep freezing process indicates that the leaf scorch symptoms result from water stress and a lack of sap warming.