Purification and serology of a Japanese isolate of barley yellow dwarf virus.
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Abstract:
A Japanese isolate of barley yellow dwarf virus (BYDV) was purified from infected plants by an improved procedure. The procedure included extraction of the virus by grinding the frozen plant tissues in a meat grinder followed by regrinding in phosphate buffer with motar and pestle for extended periods (more than 2hr) in room temperature, clarification of the sap with chloroform, concentration of the virus by polyethylene glycol, two cycles of differential centrifugation and a sucrose density gradient centrifugation. The average yield of the virus was 44μg per 100g tissues. The Japanese isolate had particle diameter of 27.3nm (2% PTA, pH 5.0) and UV absorbance spectrum with an average A260/A280 ratio of 1.71. In ELISA using the double antibody sandwich method, the virus reacted only with homologous antiserum but not with antiserum to potato leafroll virus (PLRV), and vice versa. The virus, however, reacted slightly with heterologous antisera to other luteoviruses such as PLRV, soybean dwarf and beet western yellows viruses in agar gel double diffusion tests.Keywords:
Barley yellow dwarf
Potato leafroll virus
Differential centrifugation
Luteovirus
Mottle
The Matanuska-Susitna Valley is one of the most fertile regions in Alaska for growing cool-season vegetables. Barley (Hordeum vulgare) and oat (Avena sativa) crops are also sown for animal feed and green manure. The most damaging and widely distributed viral disease of small grains worldwide is barley yellow dwarf (BYD), caused by several species from two genera in the family Luteoviridae: luteovirus (Barley yellow dwarf virus [BYDV-MAV and BYDV-PAV]) and polerovirus (Cereal yellow dwarf virus [CYDV-RPV, formerly BYDV-RPV]) and three unassigned species (BYDV-RMV, BYDV-SGV, and BYDV-GPV) (2,4). Even though barley and oat have been grown in Alaska for more than 50 years, BYD has not been documented in small grains in this region. During September 2001, barley plants with bright yellow leaves were collected from five barley fields near Palmer. Three plants from each field were assayed using a reverse transcription-polymerase chain reaction (RT-PCR) protocol targeting members of the luteoviridae (3). The resulting ≈530-bp PCR product and its restriction fragment length polymorphism (RFLP) produced by digestion with NdeII implied that plants were infected with BYDV-PAV. In September 2002, three of the five sites were surveyed again for BYDV. Two of the fields (BF-1 and BF-2) had been replanted with barley and the other (OF-3) was planted with oats. Leaf samples from 36 symptomatic barley plants from each field and 60 symptomatic oat plants were randomly collected and stored at -80°C. In 2002, in addition to RT-PCR and RFLP analyses, enzyme-linked immunosorbent assays (ELISA) using Agdia kits (Agdia, Elkhart, IN) for BYDV-PAV, CYDV-RPV, and BYDV-SGV were also performed (1). First, RT-PCR and RFLP were completed on all samples using 0.5 g of tissue. Of samples from BF-1, BF-2, and OF-3, 61, 100, and 70%, respectively, generated luteoviridae-specific fragments. The RFLP profiles from barley were all PAV-like, whereas 71% of oat samples were PAV-like, and 29% were of an unknown pattern. No bands were observed from apparently healthy field plants. ELISA (0.2 g of tissue) was performed on all PCR-positive samples, resulting in 22, 97, and 33% detection for BYDV-PAV from BF-1, BF-2, and OF-3, respectively. An additional 29% of oat samples (OF-3) tested positive for CYDV-RPV, whereas none of the barley plants tested positive. One oat plant had a mixed infection with both PAV and RPV profiles, and all oat plants with the unidentified RFLP pattern were serologically positive for RPV. No BYDV-SGV was detected in either barley or oats. The PCR assay was clearly more sensitive than ELISA, especially for plants that had mature and necrotic tissue, which were predominately found in BF-1 and OF-3. Based on these direct tests on the coat protein's nucleic acid (PCR) and serology (ELISA), it is concluded that two distinct viruses, BYDV-PAV and CYDV-RPV, were found in oats, whereas only the former was found in barley. To my knowledge, this is the first report of luteovirus and polerovirus infection in small grains in Alaska. References: (1) M. F. Clark and A. N. Adams. J. Gen. Virol. 34, 475, 1977. (2) C. J. D'Arcy and P. A. Burnett. Barley Yellow Dwarf: 40 Years of Progress. The American Phytopathological Society, St. Paul, MN, 1995. (3) N. L. Robertson and R. French. J. Gen. Virol. 72,1473, 1991. (4) M. H. V. van Regenamortel et al. Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses. Academic Press, NY, 2000.
Barley yellow dwarf
Luteovirus
Avena
Avena fatua
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Summary The concentration of potato leafroll luteovirus (PLRV) did not differ in potato plants with secondary infections grown at 15°C or 27°C. Detached leaves of plants grown at 15°C or 27°C were used as sources of PLRV for peach‐potato aphids ( Myzus persicae Sulz.) both at 15°C and 27°C. At comparable temperature during virus acquisition, aphids which fed on leaves of plants kept previously at 15°C contained more viral antigen detected by ELISA than aphids which fed on leaves of plants grown at 27°C. The aphids which acquired PLRV at 27°C contained evidently more viral antigen than those which acquired PLRV at 15°C. The greatest amount of PLRV was found in the aphids which acquired the virus at 27°C from the leaves of plants kept at 15°C. The ability of M. persicae to transmit PLRV to Physalis ftoridana Rydb. generally decreased with decrease in the amount of PLRV in vectors.
Myzus persicae
Potato leafroll virus
Luteovirus
Physalis
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SUMMARY In immunosorbent electron microscopy tests, large numbers of particles of a Scottish isolate of potato leafroll virus (PLRV) became attached to grids coated with antisera prepared to Canadian, Japanese or Swiss isolates of PLRV. Moderate numbers of particles became attached using antiserum to tobacco necrotic dwarf virus or to bean leafroll virus, smaller numbers using antiserum to soybean dwarf virus or to barley yellow dwarf virus (RPV strain) and a few particles became attached using antiserum to barley yellow dwarf virus (MAV strain) or to beet western yellows virus. A similar pattern of antigenic relationships was deduced from tests in which the binding of antibody molecules to PLRV particles exposed to different concentrations of the antisera was assessed by electron microscopy. It is concluded that these viruses should all be included in the luteovirus group and that their apparent degree of relationship to PLRV is: tobacco necrotic dwarf virus > bean leafroll virus > soybean dwarf virus > barley yellow dwarf virus (RPV strain) > barley yellow dwarf virus (MAV strain).
Luteovirus
Barley yellow dwarf
Potato leafroll virus
Strain (injury)
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Luteovirus
Barley yellow dwarf
Immunogen
Potato leafroll virus
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SUMMARY Rat monoclonal antibodies (MAbs) specific for a British F (MAV‐like) isolate of barley yellow dwarf virus (BYDV) were produced and studied. In indirect ELISA using an antiserum to BYDV‐F to trap virus from infected sap, the MAbs were shown to be specific for MAV‐like isolates of BYDV from Britain, USA and Sweden but, in this test, they did not detect PAV‐, RPV‐, SGV‐ or RMV‐ like isolates of BYDV. In similar tests using homologous antisera to trap the viruses, the MAbs did not detect BYDV‐PAV or ‐RPV or two other luteoviruses (potato leafroll and beet western yellows). One of the MAbs (MAFF 2) was partially purified from ascitic fluid, and used successfully in ELISA as a coating antibody and when conjugated to the enzyme alkaline phosphatase. Also, MAFF 2 successfully trapped BYDV‐F particles when used to coat electron microscope grids. In indirect ELISA using three MAbs (MAFF 2, MAC 91 and MAC 92) it was possible to type the three major strain groups of BYDV, viz. MAV, PAV and RPV‐like strains from Britain, USA and Europe.
Barley yellow dwarf
Luteovirus
Potato leafroll virus
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Citations (23)
Barley Yellow Dwarf (BYD) is a serious Luteovirus disease that affects small grain production worldwide. The aphid-transmitted virus (BYDV) infects practically all members of the Graminae (Poaceae) and is responsible for serious losses in cultivated species such as barley, wheat and oats. The study of BYD is complex, as it involves interactions among a vector, a plant and a virus. Hence, symptom expression is highly dependent on environmental conditions, serotypes plant genetic background and physiological stage of inoculation. Consequently, tolerance to BYDV is also difficult to study and understand. This review explores the basic biology of BYD, its symptoms, its viruses and yield losses it can cause.
Barley yellow dwarf
Luteovirus
Rhopalosiphum padi
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SUMMARY Ten murine monoclonal antibodies (MAbs) specific for a British isolate of potato leafroll luteovirus (PLRV) were produced in large quantities in ascitic fluids and their isotypes determined. All 10 MAbs reacted in indirect ELISA with intact PLRV particles but four did not react with disrupted virus particles, suggesting that these four MAbs are specific for epitopes dependent on quaternary structure. None of the MAbs gave a precipitin reaction in immunodiffusion tests. All the MAbs reacted strongly with, but failed to differentiate, 28 readily aphid-transmissible British PLRV isolates, six of which consistently caused symptoms differing in severity in indicator hosts. Two MAbs (SCR-8 and SCR-10) reacted only weakly with two other PLRV isolates, both of which were poorly transmissible by aphids. Three MAbs (SCR-6, SCR-8 and SCR-10) reacted with groundnut rosette assistor luteovirus (GRAV), but none reacted with British isolates of three other luteoviruses: carrot red leaf, beet western yellows and barley yellow dwarf (B and F isolates). Five epitopes on PLRV particles were distinguished, of which the two that were missing in the poorly aphid-transmissible isolates of PLRV and/or were shared with GRAV were both apparently dependent on quaternary structure.
Potato leafroll virus
Luteovirus
Myzus persicae
Precipitin
Closterovirus
Antigenicity
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Summary A virus obtained from accession DLP 1541 of the International Potato Center (CIP) sweet potato germplasm collection was shown to induce a curling and a white speckling of the leaves of infected plants. We propose to name it sweet potato leaf speckling virus (SPLSV). It was found in samples from 15 of 77 accessions tested and also in samples from farms at Chiclayo and Trujillo in northern Peru. SPLSV was transmissible by grafting, but not mechanically, and was transmitted in a persistent manner by Macrosiphum euphorbiae but not by Myzus persicae, Aphis gossypii or whiteflies. Isometric particles were purified from infected plants by procedures designed to purify luteoviruses. Reverse transcription and polymerase chain reaction amplification of RNA from SPLSV‐infected plants using degenerate primers designed to amplify sequences either in luteovirus RNA or in potato leafroll virus (PLRV) RNA yielded c. 500 bp and c. 600 bp DNA fragments respectively. The nucleotide sequence of these fragments encoded two polypeptides characteristic in sequence of luteovirus proteins, which suggests that SPLSV should be classified in the genus Luteovirus. The amino acid sequence of the putative coat protein was 70% identical to that of PLRV. On the basis of the coat protein sequences, SPLSV is more similar to PLRV than is any other luteovirus. However, SPLSV particles did not react with antibodies to PLRV in ISEM or ELISA tests.
Potato leafroll virus
Luteovirus
Myzus persicae
Macrosiphum euphorbiae
Aphis gossypii
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Potato leafroll virus
Luteovirus
Potato virus X
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Citations (86)
Most known luteoviruses can be placed in one of two subgroups as defined by serological relationships and genome organization (ref. 1; see Fig. 1). Subgroup I is represented by three strains of barley yellow dwarf virus (BYDV-PAV, BYDV-MAV, and BYDV-SGV). Subgroup II includes potato leaf roll virus (PLRV), beet western yellows virus, and two additional strains of BYDV (BYDV-RPV and BYDV-RMV). Soybean dwarf virus has genomic properties of both subgroups (2) and there are other luteoviruses of uncertain affinity (1). A wide range of variants exist, especially among the BYDVs. As a group, the luteoviruses infect both monocotyledonous and dicotyledonous plants, but the BYDVs apparently infect only monocots. Purification procedures are presented for isolating BYDV from a monocot and PLRV from a dicot. The procedures currently used in our laboratories are designed to overcome similar problems and so have common features, and each can be further optimized for work with specific strains or isolates (3,4). A procedure has been described that was effective in purifying six different luteoviruses representing both subgroups, and from both monocot and dicot plant tissues (5). A single procedure is presented for isolating RNA from purified virions, although several methods of RNA extraction are applicable (6).
Luteovirus
Barley yellow dwarf
Potato leafroll virus
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