Involvement of Volatile Substances in Systemic Resistance of Barley Against Erysiphe graminis f. sp. hordei Induced by Pruning of Leaves

Horsegram yellow mosaic disease was shown to be caused by a geminivirus; horsegram yellow mosaic virus (HYMV). The virus could not be transmitted by mechanical sap inoculation. Leaf dip and purified virus preparations showed geminate virus particles, measuring 15-18 * 30 nm. An antiserum for HYMV was produced and in enzyme-linked immunosorbent assay (ELISA) and immunosorbent electron microscopy (ISEM) tests HYMV was detected in leaf extracts of fieldinfected bambara groundnut, french bean, groundnut, limabean, mungbean, pigeonpea and soybean showing yellow mosaic symptoms. Bemisia tabaci fed on purified HYMV through a parafilm membrane transmitted the virus to all the hosts listed above but not to Ageratum conyzoides, okra, cassava, cowpea, Croton bonplandianus, Lab-lab purpureus, Malvastrum coromandalianum and tomato. No reaction was obtained in ELISA and ISEM tests between HYMV antibodies and extracts of plants diseased by whitefly-transmitted agents in India such as A. conyzoides yellow mosaic, okra yellow vein mosaic, C. bonplandianus, yellow vein mosaic, M. coromandalianum yellow vein mosaic, tomato leaf curl and cassava mosaic. HYMV was also not found to be related serologically to bean golden mosaic, virus.     

Purification and partial characterisation of beet mild yellowing virus and its serological detection in plants and aphids

Beet mild yellowing virus (BMW) was reversibly precipitated at temperatures below about 5°C and this property was used as a final step in a purification procedure which yielded about 1 mg virus/kg tissue. Purified virus was infective and had an A200/A280 ratio of about 1–8. BMW particles were isometric with a diameter of 26 nm, sedimented at 116 S, had a buoyant density in caesium chloride of 1.42 g/cm³ and a coat protein mol. wt of 25 400. An antiserum to BMW had a titre in immunodiffusion tests of 1/256 and was used in immunodiffusion tests, immunospecific electron microscopy (ISEM) and enzyme-linked immunosorbent assay to demonstrate a close serological relationship between BMW and beet western yellows virus. BMW was readily detected by ISEM in plants and also in aphid vectors after treatment of aphid extracts with a chloroform:butanol mixture.     

Purification and particle properties of groundnut rosette assistor virus and production of a specific antiserum*

Purified preparations of the luteovirus, groundnut rosette assistor virus (GRAV), were made by treatment of groundnut leaf extracts with cellulase, followed by sucrose density gradient centrifugation. Yields of virus particles were about 0·5-1·0 mg/kg leaf material. The preparations contained isometric particles c. 28 nm in diameter with a sedimentation coefficient (s_{20, w}) of 115 S, a buoyant density in Cs₂SO₄ of 1·34 g/cm³, and A₂₆₀/A₂₈₀ of 1·86. The particles contained a single species of nucleic acid (presumably RNA), of mol. wt 2·09 × 10⁶and with no detectable polyadenylate sequence, and a single protein species, of mol. wt 24 × 10³. An antiserum produced in a rabbit had a titre of 1/256 in gel diffusion tests and detected GRAV in leaf extracts by ELISA. GRAV particles reacted in F(ab')₂-ELISA and immunosorbent electron microscopy (ISEM) tests with antisera to bean leaf roll, potato leafroll and tobacco necrotic dwarf luteoviruses, but did not react with antisera to carrot red leaf luteovirus.     

An Isolate of Cucumber Mosaic Virus from Fluted Pumpkin in Nigeria

The properties of a virus isolated from fluted pumpkin (Telfairia occidentalis) was investigated. It produced symptoms in some members of the Solanaceae and Leguminosae, and was transmitted nonpersistently by the aphid, Aphis spiraecola. On the basis of these alone, it is distinct from another previously described virus, Telfairia mosaic virus, which neither caused symptoms in members of these families nor was it transmitted by insects. Furthermore, the virus in crude sap or purified preparations reacted with antiserum to cucumber mosaic virus (CMV), but not with antisera to several common viruses in Nigeria. Electron microscopic examination revealed isometric particles of 29 × 1 nm in diameter. These properties confirmed that the virus is an isolate of CMV, and closely resembles the Y-strain in causing systemic mosaic symptoms in Vigna unguiculata. From infectivity and pathogenicity tests, it is concluded that it is the main cause of mosaic disease in fluted pumpkin.     

Purification and some particle properties of anthriscus yellows virus, a phloem-limited semi-persistent aphid-borne virus

Anthriscus yellows virus (AYV), a phloem-limited virus transmitted in the semi-persistent manner by the aphid Cavariella aegopodii, was purified by treatment of leaf extracts with cellulasc, followed by differential and sucrose density gradient centrifugation. ‘The preparations contained isometric particles c. 29 nm in diameter which were unstable unless stored in buffer at pH 8.0 containing 1 mM CaCl₂,. The particles sedimented as two components, ’full‘ nucleoprotein particles with A₂₆₀/A₂₈₀= 1.83 containing about 42% nucleic acid, and ’empty‘ protein shells with A₂₆₀,/A₂₈₀= 0.73; their buoyant densities in CsCl solutions were 1.52 and 1.27 g/cm³. Respectively. Yields of ihe nircleoprotein particles were c. 1.75 mg/kg leaf tissue. The particles contained a single species of RNA, of mol. wt 3.6 × 10 “(10 000 nucleotides). Particle protein preparations contained four electrophoretic species, of mol. wt (× 10³) 35.0, 28.3, 23.3 and 22.3.C. aegopodii did not transmit AYV from purified preparations. A rabbit injected with AYV preparations produced antibodies that coated AYV particles in electron microscope tests, but gave variable reactions in immunosorbent electron microscopy (ISEM), depending on the composition of the medium. No reactions were obtained in enzyme-linked inimunosorbent asjay (ELISA). No serological relationship was detected in ISEM between AYV and any of 10 viruses that resembled it in one or more properties.     

Purification and properties of Australian lucerne latent virus, a seed-borne virus having affinities with nepoviruses

An isolate of Australian lucerne latent virus (ALLV) from lucerne in New Zealand was mechanically transmitted to a few herbaceous hosts. It induced diagnostic symptoms in several species of the Chenopodiaceae, but was symptomless in most other hosts including lucerne and Trifolium subterraneum. It was seed transmitted in lucerne. When assayed to Chenopodium quinoa, infective C. quinoa sap lost infectivity after diluting to 10^-4, heating for 10 min at 55°C and storage for 4 days at 4°C. ALLV was purified from infected C. quinoa or pea plants by extracting sap in 0.1 m borate buffer (pH 7) containing 0.2% 2-mercaptoethanol and clarifying with 15% bentonite suspension, high and low speed centrifugation and sucrose density gradient centrifugation. Purified virus preparations contained isometric particles about 25 nm in diameter and sedimented as three virus components with sedimentation coefficients (s_20-w⁰) of 56 S, 128 S and 133 S. The 56 S component appeared to consist of nucleic acid-free protein shells. Polyacrylamide gel electrophoresis of virus preparations showed that ALLV contained a single protein species of mol. wt 55 000 and two RNA species of mol. wt 2.1 × 10⁶ and 2.4 × 10⁶. An antiserum to ALLV had an homologous titre of 1/256 to purified virus but failed to detect ALLV in infective sap of C. quinoa, pea or lucerne. Purified ALLV failed to react to antisera to 28 distinct isometric plant viruses including those to 10 nepoviruses.     

Purification and serology of the W strain of cucumber mosaic virus

During studies on the purification of cucumber mosaic virus (strain W) it was found that preparations were most infective and stable when made from tobacco leaves (10–12 days after inoculation) homogenized in phosphate buffer containing EDTA and thioglycollic acid and clarified with diethyl ether. The preparations were further purified by centrifugation in sucrose density gradients containing EDTA at pH 9.0 and were then stable at 2 °C for > 100 days. When mounted in neutralized ammonium molybdate they were shown to consist of predominantly intact particles. In tube and ring precipitin tests and in agar gel-diffusion tests, specific precipitation with homologous antiserum occurred only in media containing alkaline adjusted solutions (ammonium molybdate and dipotassium hydrogen phosphate).     

Melon rugose mosaic virus, the cause of a disease of watermelon and sweet melon

A mechanically transmissible virus with isometric particles c. 32 nm in diameter, was isolated from infected watermelons and sweet melons in the People's Democratic Republic of Yemen. Purified virus preparations contained two major sedimenting components with sedimentation coefficients of 61S and 117S. In isopycn ic centrifugation in CsCl the particles formed a single band of buoyant density 1.39 g cm^-3. Preparations of virus particles comprised of a single polypeptide of mol. wt c. 22 000 and ssRNA of mol. wt 2.1 × 10⁶. The virus was serologically related to three of six subgroups of tymoviruses tested. The name melon rugose mosaic virus is proposed for this newly described virus.     

Purification and Characterization of Indian Cassava Mosaic Virus

The Indian cassava mosaic virus (ICMV) was transmitted by the whitefly Bemisia tabaci and sap inoculation. ICMV was purified from cassava and from systemically infected Nicotiana benthamiana leaves. Geminate particles of 16–18 × 30 nm in size were observed by electron microscopy. The particles contained a single major protein of an estimated molecular weight of 34,000. Specific antiserum trapped geminate particles from the extracts of infected cassava and N. benthamiana plants in ISEM test. The virus was detected in crude extracts of infected cassava, ceara rubber, TV. benthamiana and N. tabacum cv. Jayasri plants by ELISA. ICMV appeared serologically related to the gemini viruses of Acalypha yellow mosaic, bhendi yellow vein mosaic, Croton yellow vein mosaic, Dolichos yellow mosaic, horsegram yellow mosaic, Malvastrum yellow vein mosaic and tobacco leaf curl.     

Properties of a host range and coat protein variant of broad bean true mosaic comovirus from Vicia faba

Unlike other described isolates of broad bean true mosaic comovirus (BBTMV), a variant, code name SB, infected some non-leguminous plant species and, in N. benthamiana, induced systemic mottling and puckering of the leaves. However, like other described BBTMV isolates, purified SB particle preparations contained isometric particles c. 28 nm in diameter that sedimented as two nucleoprotein components with S_{20, w} values of 90S and 109S; some preparations occasionally contained a component of c. 50S. Virus particles contained two ssRNA species which, when denatured in glyoxal, had estimated M_T values of 2.1 × 10⁶ and 1.3 × 10⁶ and co-electrophoresed with cowpea mosaic virus RNA-1 and RNA-2 respectively. Isolate SB was serologically indistinguishable from British and German isolates of BBTMV. However, SB virus particles contained a major polypeptide (L) of M_r between c. 31 000 and up to three minor ones (S) or M_r between c. 20 000 and 24 000. This contrasts with protein preparations from other BBTMV isolates that typically contain only two polypeptides of M_r c. 37 000 (L) and 21 000 (S). Following isopycnic centrifugation in CsCl, SB particles purified from pea separated into two major components with densities of 1.39 and 1.44 g cm^-3 and a minor component of estimated density 1.43 g cm^-3. In Cs₂SO₄, virus preparations separated into three major components with densities of 1.30, 1.32 and 1.36 g cm^-3 and a minor one of density 1.27 g cm^-3. In CsCl isopycnic gradients, SB particles purified from TV. benthamiana separated into two components with densities of 1.38 and 1.43 g cm^-3. During immuno-electrophoresis in agarose gels, freshly prepared virus and preparations stored for up to 4 days at 4°C contained a single component that migrated rapidly to the anode, whereas similar preparations of an English isolate of BBTMV migrated as a single component that moved only slowly toward the anode but which, within 48 h, contained an additional component with a migration rate similar to that of isolate SB. Isolate SB is therefore a host range variant of BBTMV which, in comparison with previously described isolates of BBTMV, has an increased negative charge of its particles prior to any appreciable degradation of its S protein, and S protein that is degraded less rapidly. These features probably account for the anomalies observed in isopycnic centrifugation.     

Partial purification and serological relationship of three strains of bean yellow mosaic virus

Three biologically distinct strains of bean yellow mosaic virus (BYMV^S, BYMV¹ PV-2) were partially purified by centrifugation at relatively low g forces. Serologically these strains appeared to be distinct from each other but were related.     

The serological relationships and some other properties of isolates of broad bean wilt virus from faba bean and pea in China

An isolate (N15) of broad bean wilt virus (BB W V) from faba bean in China was compared with some other isolates and strains including the nasturtium ringspot strain (NRSV, BBWV serotype I), parsley virus 3 (PV3, serotype I) and BBWV isolate PV131 (serotype II). In host range studies, N15 infected 12 of 14 species, including soybean and spinach. It was purified from Chenopodium quinoa and pea by a method that yielded up to 8mg/100g tissue. By the same method, NRSV yielded up to 4mg/100 g. Purified preparations of N15 and NRSV contained isometric particles c. 26 nm in diameter which sedimented as three components, N15 at 62, 93 and 117 S, and NRSV at 60, 91 and 116 S. In immunodiffusion tests using antisera to N15 and NRSV, N15 was distinguishable from NRSV but indistinguishable from PV131. In ISEM tests, many more particles of N15 and NRSV were trapped by homologous than by heterologous antiserum; in decoration tests, much antibody attached to homologous particles but none to heterologous particles. In DAS ELISA using N15 antiserum, N15 and six other Chinese faba bean or pea isolates, and a Chinese spinach isolate, were readily detected and were indistinguishable from each other and from PV131; unlike NRSV and PV3, none of the Chinese isolates, nor PV131, was detected using NRSV antiserum. These results indicate that the Chinese isolates belong to BBWV serotype II group.     

Serological Studies on Mung Bean Yellow Mosaic Virus

Particles of mung bean yellow mosaic virus (MYMV) were purified by a method that yields up to 3 mg per kg of systemically infected Phaseolus vulgaris“Top Crop” and used to prepare antiserum. MYMV antiserum prepared gave a single precipitin line and had a titre of 1/512 with homologous virus in gel double-diffusion tests. MYMV was shown to be serologically related to other whitefly-transmitted viruses, bean golden mosaic virus, tobacco leaf curl virus and cassava latent virus.     

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/*.     

Purification, Properties and Serology of Strawberry Mild Yellow-Edge Virus

Oregon isolate My-18 of strawberry mild yellow-edge virus (SMYEV) was purified by comminution in liquid nitrogen, extraction in 0.1 M phosphate, 0.01 M DIECA, 1 % thioglycollic acid (pH 7.0) and differential and rate-zonal density gradient (dg) centrifugation. The resulting ultraviolet-absorbing dg band (A_{254 nm}), not seen in healthy control preparations, contained isometric, 23 mm-diameter, virus-like particles. The partially purified MY-18 virus was not transmitted to Fragaria vesca by means of membrane-fed or injected Chaetosiphon fragaefolii. MY-18 has an in vivo thermal inactivation point between 45 and 50 °C as determined by feeding C. fragaefolii on detached leaves that had been immersed in water for 10 min at various temperatures. In ELISA, rabbit antisera against MY-18 differentiated between partially purified preparations from root and leaf tissue and between crude root but not crude leaf extracts from healthy and MY-18-infected Fragaria. Our data support the generally held hypothesis that SMYEV is a luteovirus. However, comparative ISEM and ELISA tests failed to reveal any serological releationship between MY-18 and potato leafroll, beet western yellows, legume yellows, pea leafroll, or tobacco necrotic dwarf viruses.     

Purification and Characterization of a Jordanian Isolate of Watermelon Mosaic Virus

The Jordanian isolate of watermelon mosaic virus-2 (WMV-2Jo) was purified from infected Cucurbita pepo cv. Top Capi by extraction in 0.05 M sodium citrate buffer containing 0.01 M sodium diethyl dithiocarbamate and 0.01 M cysteine hydrochloride (0.01 M D + C), clarification with chloroform and n-butanol, sedimentation by ultracentrifugation, and rate-zonal centrifugation in 10–40% sucrose gradient. The purified virus had an ultraviolet absorption spectrum typical of a nucleoprotein with a low nucleic acid content. Homologous antiserum had a titre of 1: 256, as determined by the ring interface test. Electron microscopy of negatively stained purified virus revealed flexuous particles with a normal length of 750 nm. Cytoplasmic spindle-shaped inclusions were readily detectable in infected epidermal cells under the light microscope. Thin sections of infected tissue revealed the presence of laminated aggregates, pinwheel and scroll inclusions. The virus reacted with antisera prepared to the Florida strain of WMV-2 and zucchini yellow mosaic virus in a sodium dodecyl sulfate (SDS) agar gel diffusion test. Using the Derrick-decoration combined technique of immune electron microscopy, the virus reacted strongly with the homologous antiserum and zucchini yellow mosaic virus antiserum, but less with antiserum prepared to the Florida strain of WMV-2.     

Purification, serological relationships and some characteristics of plum line-pattern virus

Plum line-pattern virus (PLV) was purified by homogenizing inoculated leaves of Nicotiana megalosiphon in 0·02 M phosphate buffer, pH 8·0 (1·5 ml/g leaf), containing 0·02 M 2-mercaptoethanol. The homogenate was centrifuged at low speed and the supernatant liquid was clarified by adjusting the pH to 4·8 with 0·1 M citric acid. The green coagulum was removed by centri-fugation and the extract adjusted to pH 6·5. After concentrating the virus by high-speed centrifugation, remaining host protein was precipitated with the gamma-globulin fraction of antiserum to N. megalosiphon protein. Purification was completed with two cycles of high- and low-speed centrifugation. Purified PLV had an A₂₆₀/A₂₈₀ ratio of c. 1·7 and formed two zones when centrifuged in density gradients at pH 6·0–7·0. The virus was about 30 mμ in diameter in negatively stained preparations. The particles were easily disrupted. PLV was closely serologically related to cultures of plum line-pattern virus from other areas, but no relationship was found to apple mosaic, Prunus necrotic ringspot or prune dwarf viruses, or to a plum line-pattern virus from Denmark.     

Purification and Serology of the Iranian Maize Mosaic Rhabdovirus1)

Leaves of maize infected with the Iranian maize mosaic rhabdovirus (IMMRV) were homogenized in 0.1 M citrate-0.04 M Na₂SO₃ buffer, pH 5.4, containing 10 % sucrose and the extract was subjected to low speed and high speed centrifugation followed by resuspension in 0.05 M potassium phosphate buffer, pH 7.2, containing 10 % sucrose. Partially purified preparation was obtained by density-gradient centrifugation, removal of the virus zones and their concentration by high speed centrifugation. Two virus specific bands were observed in density-gradient columns. An antiserum with a titer of 128 was prepared by injecting partially purified virus into rabbits. In agar-gel-diffusion tests, the antiserum produced one or two precipitin lines against diseased maize extract but none against healthy maize extract. IMMRV was not related to barley yellow striate mosaic (BYSMV), cereal chlorotic mottle (CCMV), Cynodon chlorotic streak (CCSV), Festuca leaf streak, and maize mosaic (MMV) viruses as well as to two unidentified rhabdoviruses occurring in wheat and Bermuda grass in the vicinity of Shiraz, when these viruses were tested against IMMRV antiserum in agar-gel-diffusion and enzyme-linked immunosorbent assay. Likewise, IMMRV did not react with antisera to BYSMV, CCMV, CCSV and MMV in agar-gel-diffusion tests. IMMRV appears to be different from most reported rhabdoviruses of cereals.     

Some properties of a previously undescribed virus from cassava: Cassava American Latent Virus

A virus with spherical particles c. 28 nm in diameter was sap-transmitted from different cassava (Manihot esculenta) cultivars to a limited range of species in the families Chenopodiaceae and Solanaceae. Cassava seedlings infected by inoculation with sap or with purified virus preparations did not show any symptom, although the virus was readily detected by ELISA or by further inoculations. Leaf extracts from infected Nicotiana benthamiana were infective after dilution of 10^--³but not 10^--⁴, and after heating for 10 min at 70°C, but not at 72°C. The virus was purified from N. benthamiana, N. clevelandii or from cassava. On sucrose gradients, the virus particles sediment as three components all containing a protein of mol. wt c. 57000. The genome of the virus is composed of two RNAs of mol. wt c. 2.54 times 10⁶(RNA-1) and 1.44 times 10⁶(RNA-2). RNA-2 was detected in the middle and the bottom nucleoprotein components, and RNA-1 only in the bottom component. An antiserum prepared to purified virus particles was used to readily detect the virus in cassava and other host plants by ELISA and by ISEM. No serological relationship was shown between this virus and eight nepoviruses, including the recently described cassava green mottle nepovirus infecting cassava in the Solomon Islands (Lennon, Aiton & Harrison, 1987). The virus described here is the first nepovirus isolated from cassava in South America, and is named cassava American latent virus.     

Narcissus mosaic virus

Narcissus mosaic virus (NMV) is widespread in British crops of trumpet, large-cupped and double daffodils, but was not found in Narcissus jonquilla or N. tazzeta. Many commercial daffodil cultivars seem totally infected, and roguing or selection is therefore impracticable. Strict precautions by breeders and raisers to prevent infection of new cultivars is recommended. Healthy daffodil seedlings were readily infected with NMV by mechanical inoculation, but the virus was not detected in them until 17 months after inoculation, when a mild mosaic appeared. NMV infected twenty-eight of fifty-three inoculated plant species; only five (Nicotiana clevelandii, Gomphrena globosa, Medicago sativa, Trifolium campestre and T. incarnatum) were infected systemically, and NMV was cultured in these and assayed in Chenopodium amaranticolor and Tetragonia expansa. The virus was not transmitted to and from G. globosa or N. clevelandii by three aphid species, or through the seeds of Narcissus, G. globosa and N. clevelandii but was transmitted by handling. G. globosa sap was infective at a dilution of 10 ^-5 but not at 10^-6, when heated for 10 min. at 70° C. but not at 75° C, and after 12 weeks at 18° C, or 36 weeks at 0–4° C. NMV withstood freezing in infected leaves and sap, and purified preparations and freeze-dried sap remained infective for over 2 years. NMV was precipitated without inactivation by ammonium sulphate (313 g./l.) but was better purified by differential centrifugation of phosphate-buffer extracts treated with n-butanol. Such virus preparations from G. globosa, N. clevelandii, C. amaranticolor and T. expansa were highly infective, serologically active, produced a specific light-scattering zone when centrifuged in density-gradients and contained numerous unaggregated particles with a commonest length of 548–568 mμ. Antisera prepared in rabbits had precipitin tube titres of 1/4096. NMV was detected in three experimental hosts but not in narcissus sap. Unlike some viruses with elongated particles, NMV precipitates with antiserum in agar-gel. Purified preparations reacted with antiserum to a Dutch isolate of NMV but not with antisera to seven other viruses having similar particles and in vitro properties, or to narcissus yellow stripe virus.