Some properties of chrysanthemum stunt, a virus with the characteristics of an uncoated ribonucleic acid

Grafting to virus-free Mistletoe chrysanthemums was the most reliable method of detecting the chrysanthemum stunt agent, but specific light and temperature conditions were required for the diagnostic ‘measles’ symptoms to develop. Although stunt agent was highly infectious, leaf-rubbing inoculations with chrysanthemum sap gave erratic results. Colorimetric and electrophoretic tests were unreliable for indexing chrysanthemums. Stunt agent infected eight of twenty-nine species in the family Compositae, but none of 116 species in forty-seven other families. Stunt spread rapidly by foliage contact and by handling plants, but dipping the hands in 2% trisodium orthophosphate when handling plants increased the amount of spread. Stunt agent was not transmitted by four species of aphids, the glasshouse redspider mite, dodder (Cuscuta campestris) or through chrysanthemum seed. Stunt agent withstood 10 min at c. 98 °C and dilution to 10^-4, was not pelleted by ultracentrifugation, and was inactivated by RNase in weak, but not strong, buffer, suggestive of an uncoated RNA ‘viroid’. Partially purified preparations were made by homogenizing frozen chrysanthemum leaves in 0.5 m phosphate buffer with antioxidant at c. 2 °C, and clarification by n-but-anol and chloroform, followed by centrifugation. Highly infective RNA was precipitated from the supernant fluid by 2.5 vol. cold ethanol, and resuspended in a small volume of buffer. The u.v. absorption spectra of infective preparations and the u.v. absorbance profiles of density-gradients, were very similar to those of preparations from healthy chrysanthemum. Infective partially purified preparations of stunt agent withstood exposure to 2% formaldehyde or tri-sodium orthophosphate, u.v. irradiation, and sonication. Stunt preparations contained no virus particles recognizable by electron microscopy, gave no distinct peak on analytical ultracentrifugation, and did not consistently contain any specific antigen. Although similar to the ‘viroids’ potato spindle tuber and citrus exocortis, stunt agent did not infect Citrus limon, Gynura aurantiaca or tomato.     

Stabilization, culture and some properties of tulip halo necrosis virus

Tulip halo necrosis virus, obtained from tulips with leaf necrosis, is very labile in crude sap but can be transmitted consistently by inoculating Nicotiana clevelandii plants with extracts made in pH 8 phosphate buffer containing a stabilizing agent such as 0.2M 2-mercaptoethanol or 0.01M dithiothreitol. Of the fifteen species in five families of Angiosperms infected by inoculation with sap, few are suitable as sources of inoculum. Cultures of the virus can be maintained in Nicotiana clevelandii kept at 14 or 18d?C but not at 21d?C. Infectivity can be assayed in Chenopodium quinoa, in which necrotic local lesions are produced. Stabilized extracts of leaves were infective at a dilution of 1/16 but rarely at 1/32, and infectivity decreased disproportionately with dilution. Infectivity of all extracts was abolished in 10 min at 50d?C and of some at 45d?C, but survived when extracts were clarified using diethyl ether or trichlorotrifluoroethane. The virus was not transmitted by the aphid Myzus persicae.     

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.     

植物组织粗汁液中的番木瓜环斑病毒的ELISA检测技术

本研究建立和改进了检测番木瓜和西葫芦组织粗汁液里的番木瓜环斑病毒（ＰＲＶ）的ＤＡＣ－ＥＬＩＳＡ法和Ｄｏｔ－ＥＬＩＳＡ法。用不同的ＥＬＩＳＡ方法来检测不同寄主植物粗汁液里的ＰＲＶ,其所用的合适的制备粗汁液的缓冲液是不同的。用ＤＡＣ－ＥＬＩＳＡ法检测西葫芦粗汁液时,以０．５ｍｏｌ／Ｌ磷酸盐缓冲液（ｐＨ７．５,内含０．１ｍｏｌ／Ｌ乙二胺四乙酸二钠）为宜;而检测番木瓜粗汁液时,则还要加入０．２５ｍｏｌ／Ｌ脲。用Ｄｏｔ－ＥＬＩＳＡ法检测时,在上述磷酸盐缓冲液中加入２％聚乙烯吡咯烷铜能提高对西葫芦粗汁液的检测效果。应用合适的制备粗汁液的缓冲液,ＤＡＣ－ＥＬＩＳＡ法和Ｄｏｔ－ＥＬＩＳＡ法的灵敏度分别提高到１／４０９６和１／１０２４（稀释度）。本研究还表明,影响ＤＡＣ－ＥＬＩＳＡ法的定过测定的主要因素是粗汁液的稀释度和包被液（０．０５ｍｏｌ／Ｌ碳酸盐缓冲液,ｐＨ９．６）的用过。在较高粗汁液稀释度和包被液的用量相同时,粗汁液里的病毒含量与ＤＡＣ－ＥＬＩＳＡ法的ＯＤ４９２ｎｍ值呈真实的线性关系。     

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.     

Transgenic resistance to Mirafiori lettuce virus in lettuce carrying inverted repeats of the viral coat protein gene

Mirafiori lettuce virus (MiLV), a plant RNA virus belonging to the genus Ophiovirus, is considered to be a causal agent of lettuce big-vein disease. In this study, inverted repeats of a fragment of the coat protein (CP) gene of MiLV in a binary vector pBI121 were transferred via Agrobacterium tumefaciens-mediated transformation into lettuce (Lactuca sativa L.) in order to generate MiLV-resistant lettuce. Forty T₁ lines were analyzed for resistance to MiLV by detecting MiLV in leaves, and two lines (lines 408 and 495) were selected as resistant to MiLV. Both lines were susceptible to Lettuce big-vein associated virus (LBVaV), and line 495 showed higher resistance to MiLV than line 408. Further analysis indicated that line 495 showed resistance to big-vein symptoms expression. Small interfering RNA (siRNA) molecules derived from the transgene were detected in plants of line 495. MiLV was detected in roots but not in leaves of line 495 plants after MiLV inoculation, suggesting that resistance to MiLV is less effective in roots than in leaves.     

Arracacha virus A, a newly recognised virus infecting arracacha (Arracacia xanthorrhiza; Umbelliferae) in the Peruvian Andes

Arracacha virus A (AVA), a previously undescribed virus, is common in arracacha (Arracacia xanthorrhiza; Umbelliferae) in the Huanuco region of the Peruvian Andes. AVA was not transmitted by Myzus persicae, but was transmitted by inoculation of sap to 38 species from 10 families out of 63 species from 12 families tested. AVA was best propagated and assayed in Chenopodium quinoa and Nicotiana clevelandii in which it caused severe diseases. Sap from infected C. quinoa was occasionally infective after dilution to 10^-4 but not 10^-5, after 10 min at 65 °C but not 70 °C, and after 15 days at 20 °C. In neutral phosphotungstate, AVA has isometric particles c. 26 nm in diameter with a hexagonal profile, some of which were either fully or partially penetrated by the negative stain. Up to 50–200 E₂₆₀^1cm units of purified virus was obtained from 1 kg of infected N. clevelandii leaf by extraction in 0.05 M phosphate buffer at pH 7.5 containing 0.05 M ethylene diaminetetra-acetate, and clarification with chloroform, followed by differential precipitation with ammonium sulphate and three cycles of differential centrifugation. Purified virus sedimented as three components with sedimentation coefficients (S_20w^°) of 50 S, 92 S and 125 S and E₂₆₀/E₂₈₀ ratios of 0.65, 1.50 and 1.85 respectively. At equilibrium in CsCl gradients, buoyant densities of the 50, 92 and 125 S components were 1.32, 1.45 and 1.52 g/cm³ respectively. From the sedimentation coefficients and buoyant densities, the nucleic acid contents of the 92 S and 125 S components were estimated at 30–35% and 43–44% respectively. Only the 125 S component seemed to be infective but its infectivity was greater when mixed with the 92 S component. All three components contained a single protein with a molecular weight of 53 000. AVA was not serologically related to any of 33 other morphologically similar viruses. Although the vector is unknown, its properties suggest that it is a member of the nepovirus group. The cryptogram of AVA is */*: */43–44 +*/30–35: S/S:S/*.     

柑桔碎叶病毒研究

用汁液摩擦接种方法对柑桔碎叶病毒(Citrus tatter leaf virus,CTLV)进行了进一步的生物学鉴定.结果表明,该病毒除在豇豆上引起枯斑外,还侵染克里芙兰烟(Nicotianaclevilandii)产生系统斑驳和轻花叶,侵染昆诺藜(Chenopodium quinoa)和苋色藜(C.amaranticolor)引起系统坏死和花叶,侵染鸡冠花(Celosia cristata)引起局部环斑.这些草本寄主均可作为CTLV的指示植物.在发病的柑桔叶汁液中,测得CTLV的稀释限点为10~(-5).经汁液摩擦接种,可以将CTLV从克里芙兰烟传播到木本指示植物柑桔和枳橙上.感病植物材料的组织超微结构观察结果表明:CTLV感染柑桔、枳橙、克里芙兰烟和昆诺藜均使其叶肉薄壁细胞的叶绿体出现淀粉沉积、叶绿体片层结构解体和消失;病毒在受感染的植株叶脉韧皮部细胞中紧密聚集,形成病毒结晶体;这种结构也出现在叶肉薄壁细胞中.这是关于该病毒组织病变的首次报道.用直接负染方法从感染CTLV的柑桔(枳橙)、克里芙兰烟和昆诺藜病叶中均能检查到线状病毒粒子,用改良的Deriick’s免疫电镜方法和琼脂双扩散方法测定均显示该病毒…     

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.     

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.     

Improved purification procedure and some serological and physica properties of cassava common mosaic virus from South America

Large quantities of cassava common mosaic virus (CCMV) were purified from systemically infected Nicotiana benthamiana plants. A polyclonal antiserum, with a titre of 1/128 in the tube precipitin test, was produced by immunising rabbits with purified virus. Viral antigens were detected in cassava, using both the double-antibody sandwich or plate-trapped antigen forms of enzyme-linked immunosorbent assay (ELISA). The virus reacted with antisera to the potexviruses potato virus X and tulip virus X in F(ab')₂ ELISA. As determined by ELISA, isolates of CCMV from cassava and chaya are closely serologically related to each other. Leaf extracts from infected N. benthamiana plants were infective to a dilution of 10^--⁴ but not 10^--⁵; after heating for 10 min at 65 °C but not 70 °C; and after storage at room temperature for 14 days. The virus has a sedimentation coefficient of 126 S_20,w, a single coat protein molecule of c . mol. wt 21 000, and a single-stranded RNA genome of c . mol. wt 2.0 ± 10⁶. Several dsRNA species, including the putative viral replicative form of c . mol. wt 4.1 ± 10⁶, were isolated from virus-infected cassava and N. benthamiana .     

Some properties of peanut clump, a newly discovered virus

A mechanically transmissible soil-borne virus causing peanut clump disease in Upper Volta is described. It infected mainly species of Chenopodia-ceae and was propagated in Chenopodium amaranticolor. Infectivity was lost from sap of C. amaranticolor after 10 min at 64 °C, and after dilution to 10^-5 but not io^-4. A purification procedure is described. The particles are rod-shaped and of two predominant lengths, 190 and 245 nm. The virus is not serologically related to tobacco rattle, pea early-browning, or soil-borne wheat mosaic viruses, or to a virus associated with a rhizomania-like disease of beet.     

Cowpea mild mottle, a newly recognized virus infecting cowpeas (Vigna unguiculata) in Ghana

Cowpea mild mottle virus (CMMV), a previously undescribed virus widespread in cowpeas (Vigna unguiculata) in the Eastern Region of Ghana, was seed-borne in V. unguiculata, Phaseolus vulgaris and Glycine max, but was not transmitted by twelve aphid species including Aphis craccivora, A. fabae, Acyrthosiphon pisum and Myzus persicae. CMMV was transmitted by inoculation of sap to eleven of seventeen members of the Papilionaceae causing very severe diseases in G. max and Arachis hypogaea, and to ten of fifty-one species within five of nineteen other families; it was best propagated in G. max and Nicotiana clevelandii, and assayed in Chenopodium quinoa. Sap from systemically infected G. max was infective after dilution to 10^-3 but not 10^-4, after 10 min at 65 °C but not at 70 °C, or after 4 days at 18 °C or 16 days at 2 °C. Lyophilized sap was infective after 3 years in vacuo. CMMV has straight to slightly flexuous, fragile filamentous particles, c. 13 × 650 nm which, in sap, are occasionally surrounded by a loose external spiral. About 5 mg of purified virus was obtained from 1 kg of leaf tissue of G. max or N. clevelandii by clarifying leaf extracts in 0.02 m borate buffer (pH 9.5) with chloroform, followed by two or three cycles of differential centrifugation, and density gradient centrifugation. Virus preparations had ultraviolet absorption spectra typical of a nucleoprotein containing c. 5 % nucleic acid, contained numerous particles without external spirals, which sedimented as a single component with a sedimentation coefficient (s°_{20, w}) of 165 × 4S, and contained a single polypeptide species with a molecular weight of 32000–33000. CMMV showed a distant serological relationship to carnation latent virus, but not to ten other morphologically similar viruses; it thus seems to be a distinct member of the carlavirus group, and has the cryptogram: */*:*/(5):E/E:S/*.     

Pathology, soil transmission and characterization of cymbidium ringspot, a virus from cymbidium orchids and white clover (Trifolium repens)

Two strains of a virus, designated cymbidium ringspot virus (CyRSV), were isolated from cymbidium orchids and from Trifolium repens respectively in Britain. Experimentally infected cymbidiums developed slight chlorotic ring-mottle; T. repens developed flecks and mottling in the leaves, and slight stunting. Of 101 plant species tested, the cymbidium strain infected sixty-one (thirteen systemically) in twenty-three of thirty-five families; the clover strain infected sixty-four species (eighteen systemically) in twenty-two families. Both strains were propagated in Nicotiana clevelandii and assayed in Chenopodium quinoa. CyRSV was readily transmitted by inoculation of sap, and by foliage contact between plants, but not by the aphids Myzus persicae or Acyrtho-siphon pisum, nor through seed of T. incarnatum, Phaseolus vulgaris or N. clevelandii. Highly infective virus was released into soil from roots of infected N. clevelandii, and acquired by bait seedlings planted in such soil. Similar transmission occurred when purified virus was applied to the surface of sterilized soil containing bait plants; there was no evidence for any living soil vector. The virus was eliminated from 96 % of small cuttings taken from infected N. clevelandii plants grown at 35–37 °C for 9 wk. CyRSV was still infective in sap of N. clevelandii after dilution to 10?⁵-io–⁶ (only 2 × 10^_1 in cymbidium sap), or after 10min at 85–90 °C. It survived at least 10 months at c. 20 °C and more than 12 yr at 2 °C. Lyophilized sap was highly infective after over 13 yr at laboratory temperatures under high vacuum. Purified preparations made by clarification with n-butanol, followed by differential centrifugation and exclusion chromatography on controlled-pore glass beads, contained isometric particles c. 30 nm diam., with s°_20W= 137 S, and had a buoyant density in caesium chloride of 1–36 g/ml. The A 260/A 280 ratio was 1–55, and A max(26o)/A min(242) was 1–17. The virus contained c. 15 % of single-stranded RNA of mol. wt 1–7 × 10⁶; the nucleotide base ratios were: G27'8; A24/9; C2I-3; U26-I. There was one capsid polypeptide of mol. wt 43600. The virus was a good immunogen and a strongly reacting antigen in vitro; in Immunoelectrophoresis, each strain migrated as a single antigenic component towards the cathode. The cymbidium and clover strains were serologically closely related, although spurs were produced in immunodiffusion. No serological relationship was found to forty-three other isometric viruses, including eighteen tombusvirus isolates; CyRSV nevertheless shares many properties with tombusviruses, and we assign it provisionally to this group. The cryptogram is: R/r:1:7/15:S/S:S/O.     

Augusta disease in tulip - a reassessment

In an experiment in which the roots of field-grown tulip were commonly infected with tobacco necrosis virus (TNV), Augusta disease did not develop in the year of infection or when progeny bulbs were grown in the field or glass-house. When tulip bulbs of other stocks, including grades of 11 and 12 cm circumference, were forced, the disease developed sporadically, in some instances as the result of infection with TNV from the soil in which they were planted and in others as a result of infection by bulb-borne virus. The incidence of disease produced by current year infection was increased by warming the plunge bed. Different strains of TNV were obtained from field-grown plants with Augusta disease and different strains of the virus produced the disease when inoculated to tulip. Some, but not all, naturally diseased plants contained satellite virus, which therefore does not cause or prevent disease development. The disease was produced in some plants by TNV transmitted by Olpidium brassicae, but neither a vector nor a non-vector isolate of O. brassicae completed its life cycle in tulip. However, Olpidium-like zoospores were observed in some washings of tulip roots from TNV-infested soils. TNV was not obtained from all tulip plants with necrotic leaf symptoms resembling Augusta disease. Some were infected with tomato bushy stunt virus or cucumber mosaic virus, or with another agent that was transmitted by inoculation of sap to Nicotiana clevelandii and Chenopodium quinoa, and carried by bulbs of up to 11 cm circumference.     

Arracacha virus B, a second isometric virus infecting arracacha (Arracacia xanthorrhiza; Umbelliferae) in the Peruvian Andes

Arracacha virus B (AVB), a previously undescribed virus, was found together with arracacha virus A or with a 750 nm flexous filamentous virus in arracacha (Arracacia Xanthorrhiza; Umbelliferae) growing in the Huanuco region of the Peruvian Andes. AVB was transmitted by inoculation of sap to 30 species from eight families out of 45 species from 10 families tested. It was transmitted through seed of Chenopodium quinoa but not by Myzus persicae. AVB was best propagated in C. Quinoa or Tetragonia expansa and assayed in C. quinoa, C. murale or C. amaranticolor. Sap from infeted <C. Quinoa was occasionally infective after dilution to 10^-4 but not 10^-5, after 10 min at 65 d? C but not 70 d? C, and after 12 but not 14 days at 20 d? C. In neutral phosphotungstate, AVB has isometric partilces c. 26 nm in diameter with a hexagonal profile. About 50- 150 A1 cm₂₆₀ units of purified virus were obtained from 1 kg infected C. quinoa leaf by extraction in 0.5 M phosphate buffer at pH 7.5, containing 0.05 M ethylene-daiminetetra-acetate (EDTA) and 0.2% mercaptoethanol, and clarificatin with chloroform, followed by two precipitations with polyethylene glycol and three cylces of differential centrifugation. Purified virus coefficent (Sd?_{20 w},) of 126 S and A₂₆₀/A₂₈₀ ratio of 1.80, bnut formed two isopycnic bands in CsC1 of buoyant density 1.481 and 1.492 g/cm³ with estimated nucleic acid contents of 40 and 41% respectively. AVB particles contained two proteins of mol.wt 26 000 (major component) and 20 000. AVB was not serologically related to any of 20 other morphologically similar viruses. Its properties suggest that it does not fall into any recognised group of viruses. the cryptogram of AVB is */*:*/40–41:S/S:S/*     

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.