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.     

Effects of Temperature on BYDV Concentration in Oat Plants and Virus Purification Efficiency, and Detection of a Putative Associated Satellite Virus

RPV and MAV-like serotypes of barley yellow dwarf virus (BYDV), designated R-568 and F, were found during sucrose density gradient centrifugation to suspend at 10 °C and 4 °C but to totally sediment at 15 °C and 12 °C, respectively. These properties were used to purify these serotypes, and antisera were then prepared.
Partially purified IgG from antiserum was used in immunosorbent electron microscopy (ISEM) and in enzyme-labelled immunosorbent (ELISA) tests to detect BYDV RPV-like serotypes. Using anti-BYDV R-568 polyclonal antiserum and the BYDV R-568 serotype in ISEM tests, isometric virus particles of two sizes were trapped: the 28 nm particles of BYDV R-568, and others 17 nm in diameter which may be those of a satellite virus.
The effects of temperatures on virus concentrations in oat plants infected with BYDV serotypes F and R-568 were investigated. BYDV F and R-568 concentrations in the roots and shoots were sensitive to changes in temperature between 10 °C and 25 °C. The concentrations of both viruses in the roots and shoots of infected plants could be manipulated by varying the temperature at which plants were grown. The ELISA absorbance values related to detection of F MAV-like serotypes were higher in roots and shoots of oats grown at 10 °C than for oats grown at 25 °C. Conversely, cool temperatures reduced the absorbance values for R-568 RPV-like serotype in the roots, but less significantly in the shoots.     

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.     

Detection and Comparison of some Ghanaian Isolates of Cacao Swollen Shoot Virus (CSSV) by Enzyme-Linked Immunosorbent Assay (ELISA) and Immunoelectron Microscopy (IEM) Using an Antiserum to CSSV Strain 1A

Various isolates of Cacao Swollen Shoot Virus (CSSV) were detected without difficulty in leaves of Theobroma cacao L. by ELISA and immunosorbent electron microscopy (ISEM) using an antiserum to severe strain 1A. Many isolates were detected with relatively high values at dilutions of 1:30, whereas some other isolates were hardly or not at all detected at this dilution. Strain 1A was detected at dilutions of up to 1: 2560 of crude leaf extracts. All isolates yielding high reactions seem to be serologically closely related to strain 1A. Strains of the mottle-leaf type (AD 191, AD 196, AD 7, AD 36, AD 135, Kpeve) and others were poorly detected; their relationship to strain 1A is discussed. A close correlation was found between results obtained by ELISA and ISEM.     

Purification and some properties of oat golden stripe virus

Oat golden stripe virus (OGSV) was maintained in oats by mechanical inoculation and purified by extraction of leaves in borate buffer, two cycles of centrifugation through sucrose cushions and isopycnic centrifugation in CsCl. An antiserum with a titre of 1/1024 in precipitin tests was prepared. Particle length distribution was bimodal with median values, respectively, of 150 and 300 nm from dip preparations. Measurements from immunosorbent electron microscopy (ISEM) and purified preparations showed that the particles had partially degraded during these procedures. The virus sedimented as two components of 168 S and 218 S and had a buoyant density of 1321 g cm^-3. Four isolates of OGSV reacted with the antiserum. Antiserum to members and possible members of the furovirus group were tested in ISEM decoration tests and in ELISA. OGSV was related to soil-borne wheat mosaic virus but not to beet necrotic yellow vein virus, hypochoeris mosaic virus or potato mop-top 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.     

A comparison of serological techniques for detecting and identifying honeybee viruses

The sensitivity and specificity of conventional Ouchterlony gel-diffusion, immuno-osmoelectrophoresis (IO), immune serum electron microscopy (ISEM), “decoration,” radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA) tests for detecting black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), Kashmir bee virus (KBV), and sacbrood virus (SBV) particles in extracts of diseased honeybees were compared. A “slow” ISEM method detected virus particles in extracts of individuals or groups of individuals diluted to 10^?3 and 10^?4, respectively, whereas the IO method and a “fast” ISEM method using protein A were one-tenth as sensitive, and Ouchterlony gel-diffusion tests were only one-thousandth as sensitive. Using the antibody “decoration” technique, mixtures of serologically unrelated virus particles could be resolved. RIA and ELISA were found to be one thousand times more sensitive than ISEM in detecting the particles of BQCV, CBPV, KBV, and SBV; however, nonspecific reactions occurred when using RIA with very dilute particle suspensions, and this made dilution endpoints difficult to assess, but this did not occur when using the ELISA method. There was little difference in the effectiveness of rabbit or hen antisera in the tests, except when protein A was used as it does not combine with hen antibodies.     

Detection of a Luteovirus in Groundnut Rosette Diseased Groundnuts (Arachis hypogaea) by Enzyme-linked Immunosorbent Assay and Immunoelectron Microscopy

In groundnut rosette diseased groundnut plants collected near Zaria, Nigeria, a luteovirus was detected by ELISA and ISEM. In ELISA only beet western yellows virus antiserum reacted, while in ISEM luteovirus particles were trapped by antisera beet western yellows virus, potato leafroll virus, pea leafroll virus and barley yellow dwarf virus. The data are in agreement with the interpretation that the assistor of groundnut rosette virus is possibly a member of the luteovirus group.     

Immunological detection of bean common mosaic virus in French bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) leaves

Bean common mosaic potyvirus (BCMV) is an important seed borne pathogen of French bean. Differential inoculation with bean common mosaic virus at cotylodonary trifoliate leaf stage and pre-flowering stage of crop growth revealed that cotyledonary leaf infection favored maximum disease expression. Under immunosorbent electron microscopy (ISEM) the virus particles of filamentous structure having a diameter of 750 nm (l) and 15 nm (w) were observed. These particles gave positive precipitin tests with polyclonal antiserum of Potato virus Y.     

Studies on a Nigerian isolate of banana streak badnavirus: I. Purification and enzyme linked immunosorbent assay

A Nigerian isolate of banana streak badnavirus (BSV) was purified and a polyclonal antiserum was produced in mice. The antiserum titre was between 1:10 000 and 1:40 000 in enzyme linked immunosorbent assay (ELISA), and showed a good specificity to BSV antigens. Comparative tests were carried out to determine the sensitivity and reliability of BSV antigen detection by double antibody sandwich (DAS)-ELISA, triple antibody sandwich (TAS)-ELISA, antigen coated plate (ACP)-ELISA, and protein-A coated antibody sandwich (PAS)-ELISA. TAS-ELISA using rabbit polyclonal antiserum to trap BSV and mouse polyclonal antiserum to detect the virus particles, was more sensitive than ACP-ELISA and PAS-ELISA and detected BSV in plant extracts from both symptomatic and some asymptomatic plants. However, immunosorbent electron microscopy detected more BSV-infected plants from asymptomatic plant samples than did TAS-ELISA. Results of this study showed that detection of BSV antigens in sap extracts by TAS-ELISA was most efficient with symptomatic tissues which occurred most frequently in the ‘cool rainy’ season. This suggests that for more reliable BSV-indexing of field samples, tissue sampling should be done during the rainy season when most BSV-infected plants express severe symptoms.     

Comparison of Polyclonal Antisera in the Detection of Tomato Spotted Wilt Virus Using the Double Antibody Sandwich and Cocktail ELISA

Different polyclonal antisera and enzyme-linked immunosorbent assay (ELISA) procedures have been tested for their potential to detect tomato spotted wilt virus (TSWV). The virus could efficiently be detected in high dilutions of sap from infected plants, and at low concentrations of purified virus and nucleocapsid protein preparations in the cocktail ELISA and the double antibody sandwich ELISA (DAS-ELISA). Amounts of 1 to 3 ng of virus protein still gave positive readings using purified preparations, while sap could be diluted approximately 100,000 times. Differences in the detection level were observed using nucleocapsid protein antiserum (anti-N-serum) and the antiserum against intact virus particles (anti-TSWV-serum), but both antisera showed to be powerful sera for the detection of TSWV. Using anti-N-serum, TSWV could be detected in highly diluted extracts of different hosts, and also in leaf extracts or intact tissues stored for 30 days under different conditions. These results indicate that the TSWV nucleocapsid protein remains antigenic for long periods.     

小麦黄花叶病毒的分离提纯及其血清学等特性

应用梯度离心和超速离心浓缩获得部分提纯的病毒制剂,产量约为7.45g/kg病叶提纯的病毒制剂的紫外吸收曲线呈典型的核蛋白吸收曲线,OD260/OD242和OD260/OD280的比值分别为1.24和1.38。病毒粒子呈线状,宽13—14nm,长度主要分布于250—300nm和550—700nm之间,1000nm以上的粒子也有检到。病毒外壳蛋白仅由一个分子量约为30Kd的亚基组成。在免疫电镜试验中、病毒粒子与日本WYMV抗血清发生强烈的血清学反应。新鲜病叶的超薄切片中可看到大量风轮体和膜状体。     

Detection of Double-Stranded RNA (DSRNA) in Crude Extracts of Virus-Infected Plants by Indirect ELISA

An antiserum against polyinosinic-polycytidylic acid (In-Cn) was used to detect double-stranded RNA (dsRNA) by indirect ELISA (ELISA-I). DsRNA from cucumber mosaic virus (CMV) and plum pox virus (PPV)-infected plants was detected using different types of extracts. The pH of the extraction buffer was very important in dsRNA detection, the highest optical density values being obtained at pH 6 or in aqueous extracts. Extracts heated at 80°C for 2 min showed increased optical density values compared with unheated extracts. DsRNA from Nicotiana benthamiana plants infected with each of six PPV isolates was readily detected by ELISA-I 50 days after inoculation. ELISA values then obtained with the In-Cn antiserum were generally higher than those obtained by double antibody sandwich ELISA using an antiserum to virus coat protein. Purified dsRNA from the same infected plants showed no visible band, but it produced a fluorescent background when analysed by polyacrylamide gel electrophoresis.     

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.     

Detection of six nepoviruses in their nematode vectors by immunosorbent electron microscopy

Particles of six nepoviruses were detected by immunosorbent electron microscopy (ISEM) in extracts of their respective vector nematodes. This technique was at least a thousand times more sensitive than conventional electron microscopy. It was also more rapid, reliable and sensitive than infectivity tests in which extracts of nematodes were inoculated to indicator plants. The viruses were detected in extracts of single nematodes, and in the roots and leaves of infector and bait plants.     

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.     

Allexivirus transmitted by eriophyid mites in garlic plants

Viruses in garlic plants (Allium sativum L.) have accumulated and evolved over generations, resulting in serious consequences for the garlic trade around the world. These viral epidemics are also known to be caused by aphids and eriophyid mites (Aceria tulipae) carrying Potyviruses, Carlaviruses, and Allexiviruses. However, little is known about viral epidemics in garlic plants caused by eriophyid mites. Therefore, this study investigated the infection of garlic plants with Allexiviruses by eriophyid mites. When healthy garlic plants were cocultured with eriophyid mites, the leaves of the garlic plants developed yellow mosaic strips and became distorted. In extracts from the eriophyid mites, Allexiviruses were observed using immunosorbent electron microscopy (ISEM). From an immunoblot analysis, coat proteins against an Allexivirus garlic-virus antiserum were clearly identified in purified extracts from collected viral-infected garlic plants, eriophyid mites, and garlic plants infected by eriophyid mites. A new strain of GarV-B was isolated and named GarV-B Korea isolate 1 (GarV-B1). The ORF1 and ORF2 in GarV-B1 contained a typical viral helicase, RNA-directed RNA polymerase (RdRp), and triple gene block protein (TGBp) for viral movement between cells. The newly identified GarV-B1 was phylogenetically grouped with GarV-C and GarV-X in the Allexivirus genus. All the results in this study demonstrated that eriophyid mites are a transmitter insect species for Allexiviruses.     

Tests for resistance to white clover mosaic virus in red and white clover

A range of red and white clover cultivars was tested for immunity to white clover mosaic virus. All plants became infected although some showed no symptoms. Enzyme-linked immunosorbent assay (ELISA) and immunosorbent electron microscopy (ISEM) revealed significant differences in virus concentration between red clover cultivars and between clones of white clover artificially infected with the virus. These differences could not, however, be related to relative yield losses.     

Comparison of nucleic acid hybridisation and other tests for detecting tobacco rattle virus in narcissus plants and potato tubers

All isolates of tobacco rattle virus (TRV) found in naturally infected narcissus leaves produced nucleoprotein particles, mostly in large concentrations but, because of antigenic diversity, less than half of the isolates were identified by immunosorbent electron microscopy (ISEM) and still fewer by enzyme-linked immunosorbent assay. All were identified by a nucleic acid hybridisation test in which DNA complementary to RNA-1 of strain PRN of TRV was allowed to react with nucleic acid extracted from leaf tissue. Spraing-affected tubers in some potato stocks yielded only NM isolates of TRV. These isolates do not produce virus particles and they were therefore not detected by ISEM. The infectivity of nucleic acid extracts from recently harvested tubers with spraing symptoms was much greater than that of extracts prepared from tubers after 8 months' storage. In other potato stocks, some spraing-affected tubers contained NM isolates and the rest contained particle-producing isolates (M isolates) of TRV. The infectivity of sap and of nucleic acid, extracted 7 months after harvest from tubers infected with M isolates, was much greater than that of nucleic acid extracted from comparable tubers infected with NM isolates. TRV was detected by nucleic acid hybridisation in extracts of almost all tubers containing either M or NM isolates, even when the tubers were not tested until 7–8 months after harvest. The probable sequence of events occurring after tubers are infected with TRV is outlined, and it is suggested that the virus will rarely become established in fields as a result of planting infected tubers.     

Highly Efficient Immunodiagnosis of Episomal Banana streak MY virus Using Polyclonal Antibodies Raised Against Recombinant Viral‐Associated Protein

Banana streak MY virus (BSMYV) is the causal agent of viral leaf streak disease of banana, which leads to considerable losses in banana production in most of the banana‐growing regions worldwide. Developing high‐throughput virus detection system is essential for managing viral diseases especially in vegetatively propagated crops like banana. In this study, viral‐associated protein (VAP) coded by ORF II of BSMYV was expressed in Escherichia coli, and polyclonal antibodies were raised against purified recombinant VAP (rVAP) fusion protein in rabbits. Specificity and sensitivity of resulting antibodies were tested in Western blot, immunosorbent electron microscopy (ISEM) and enzyme‐linked immunosorbent assays (ELISAs). In direct antigen‐coated (DAC)‐ELISA, antibodies reacted specifically to BSMYV in crude sap, up to 1 : 8000 dilutions, but not to healthy leaf extracts. Using this antiserum, an immunocapture polymerase chain reaction (IC‐PCR) assay was developed and compared with DAC‐ELISA. VAP antibody‐based IC‐PCR is highly specific and could differentiate episomal virus infection from the integrated endogenous BSV (eBSV) sequences. The recombinant antibodies were validated by testing with a large number of banana germplasm conserved in the field gene bank. Field samples collected during surveys and mother cultures used in tissue culture propagation suggest that antibodies generated against rVAP are sensitive and useful for large‐scale detection of BSMYV. To the best of our knowledge, this is the first report on the production of polyclonal antiserum against recombinant VAP of BSMYV and its suitability for serology‐based testing by ELISA and IC‐PCR. This VAP‐based immunodiagnosis can be applied in quarantine, germplasm exchange and certification programmes.