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ABSTRACT: Primus necrotic ringspot virus (PNRSV) isolates were characterised by bioassays, serotyping and restriction fragment length polymorphism (RFLP) analysis of PCR products. Based on symptoms in host trees and bioassays it was concluded that only one of the 16 tested isolates is severe. The serotyping results demonstrated that by using four different MAbs in TAS-ELISA the tested isolates could be divided into four subgroups, however, the severe isolate could not be singled out. RFLP analysis of PCR products supported the serotyping data but did not differentiate between isolates of the two main serological subgroups. A restriction map, derived from sequence analysis of the PCR products obtained from selected isolates, allowed exact location of the restriction sites within the PCR products of each isolate. A mild isolate with a unique genome structure was identified by both serological and RFLP assays. As far as we are aware, this is the first report on sub-grouping of PNRSV isolates by bioassays, serotyping with MAbs and RFLP analysis.
Annals of Applied Biology 06/2008; 135(1):395 - 400. · 2.18 Impact Factor
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ABSTRACT: The naturally polyadenylated genomic RNA of PYV served as a template for the synthesis of complementary DNA (cDNA) strand-primed with oligo-dT using the enzyme avian myeloblastosis virus reverse transcriptase. A second cDNA strand was made with DNA polymerase I and the double-stranded product (about 10 kbp) was cleaved with a Hind III restriction endonuclease. The resulting five main fragments of 800,950, 1250,2400 and 2600 bp were subsequently cloned into the Hind III site of the pBR322 plasmid of Escherichia coli. Three types of cDNA inserts (800, 950 and 2400 bp) were detected among the isolated recombinant plasmid clones. Intact untreated virus particles and phenolized virus samples hybridized with the cloned cDNA sequences. These clones hybridized with a major virus RNA band of about 10 kbp and a minor band of higher molecular weight in blots of RNA extracted from purified virus particles and fractionated in a denaturing agarose gel. The prospects of using cloned PVY sequences for the analysis of the virus genome and for virus detection are discussed.
Plant Pathology 04/2007; 35(2):178 - 184. · 2.13 Impact Factor
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ABSTRACT: Greenhouse-grown cucumber plants showed mosaic-type symptoms and irregular yellow spots on their leaves. The disease did not affect plant growth and the fruits remained symptom free. A virus having isometric particles, 30 nm in size, was isolated from the infected tissues and from recycled drainage water collected from tuff (volcanic rock) raised beds on which plants were grown. The virus was identified as a variant of cucumber leaf spot virus (CLSV) that has a host range similar but not identical to that of a previously described CLSV isolate. The overall nucleotide sequence identity between the RNAs of the Israeli isolate and the type isolate virus (accession numbers: DQ227315 and AY571334, respectively) amounts to 96%.
Annals of Applied Biology 11/2006; 149(3):313 - 316. · 2.18 Impact Factor
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ABSTRACT: Conspicuous viral symptoms were seen on Tabernaemontana divaricata, a member of the family Apocynaceae, grown in a commercial nursery, in Israel. The symptoms varied widely and included chlorotic ringspots and banding, oak-leaf patterns and mosaic. At the end of the winter, large yellow spots, which later became necrotic, appeared on fully expanded leaves. The necrotic zones later fell out, leaving “shot holes”. Preliminary analysis suggested that the disease was associated with a tobamovirus. Particles typical of a tobamovirus were observed by electron microscopy only in samples taken from symptomatic leaves. Apartial segment of the 5′-terminus of the viral RNA, which comprised of 533 bp was cloned and sequenced. Comparison of the predicted amino acid sequence with those of other tobamoviruses revealed 94% identity with Tobacco mild green mosaic virus (TMGMV) genome. Primers specific to the coat protein (CP) gene of TMGMV used in a reverse transcription-polymerase chain reaction assay (RT-PCR) gave an expected amplification product of 455 bp. The amino acid composition of the cloned CP gene, which shows a complete identity to that of TMGMV, confirmed the identity of TMGMV infecting Tabernaemontana. Polyclonal antibodies prepared against the virus and used in double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) enabled specific detection of the virus in crude sap extracted from T. divaricata and mechanically inoculated indicator plants. This is the first report of TMGMV infection in Tabernaemontana and the first incidence of the virus in Israel.
Annals of Applied Biology 06/2006; 138(2):153 - 159. · 2.18 Impact Factor
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ABSTRACT: The two most widespread biotypes of Bemisia tabaci (Gennadius) in southern Europe and the Middle East are referred to as the B and Q-type, which are morphologically indistinguishable. In this study various DNA markers have been developed, applied and compared for studying genetic diversity and distribution of the two biotypes. For developing sequence characterized amplified regions (SCAR) and cleaved amplified polymorphic sequences (CAPS) techniques, single random amplified polymorphic DNA (RAPD) fragments of B and Q biotypes, respectively, were used. The CAPS were investigated on the basis of nuclear sodium channel and the mitochondrial cytochrome oxidase I genes (mtCOI) sequences. In general, complete agreement was found between the different markers used. Analysis of field samples collected in Israel for several years, using these markers, indicated that the percentage of the Q biotype tends to increase in field populations as time progresses. This may be attributed to the resistance of the Q biotype to neonicotinoids and pyriproxyfen and the susceptibility of the B biotype to these insecticides.
Bulletin of Entomological Research 01/2006; 95(6):605-13. · 1.88 Impact Factor
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ABSTRACT: SummaryA PVY isolate which causes strong necrotic lesions in tomato was isolated and characterised. It did not infect potato and did not react with antiserum specific to the N-strain of PVY. Restriction endonuclease cleavage patterns of PCR products derived from the 5′-end of the virus genome (Hinc II and Rsa I) clearly distinguished the tomato, pepper and potato strains. Additional sequence analysis indicated that the tomato and pepper isolates were closely related, while both markedly differed from the necrotic strains of potato. Hence, it was concluded that the necrotic tomato isolate is uniquely specific for tomato.
Annals of Applied Biology 03/2005; 137(3):253 - 257. · 2.18 Impact Factor
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ABSTRACT: Prunus necrotic ringspot virus (PNRSV) was detected in almonds, plum and apricot germplasm accessions and local almond cultivars in Israel. PNRSV was widespread both in wild and cultivated almond trees and uncommon in wild apricots and plums. The possible variation among the PNRSV isolates was initially evaluated by restriction analysis of PCR products representing the CP gene with the endonuclease RsaI and followed by nucleotide sequence analysis of selected isolates. It was concluded that all 13 isolates belong to group PV96, the largest cluster of PNRSV isolates, described previously. Two PNRSV isolates, one from a plum accession and one from an almond cultivar, were found to be distinct members of group PV96 with unique nucleotide modifications not found in other documented isolates of this virus. However, no PNRSV isolate typical to a specific host and/or to the Middle East region could be identified. This study expands the body of data on variability of PNRSV isolates and highlights the importance of assessing the virus status of germplasm collections by applying reliable diagnostic and differentiating methods.
Annals of Applied Biology 03/2004; 144(2):229 - 236. · 2.18 Impact Factor
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ABSTRACT: Transgenic tomato plants carrying a truncated replication associated protein (T-Rep) gene of the mild strain of Tomato yellow leaf curl virus-Israel (TYLCV-Is [Mild]) were prepared. The transgene encoding the first 129 amino acids of Rep conferred resistance only against the virus strain from which it was derived, while these plants were susceptible to the severe strain of TYLCV-Is. This strain-specific effect may be the result of high sequence divergence within the N-terminal domains of the Rep genes of the two virus isolates which share a mere 78% sequence identity at the nucleotide level and 77% at the amino acid level. Although the transgenic tomato plants were totally resistant to whitefly inoculation with the mild strain of TYLCV-Is, agroinoculation with the same virus strain resulted in variable resistance responses in the tested plants: while 21% of plants were totally immune to the virus, 33% were susceptible and 46% expressed a wide range of intermediate resistance characteristics. The applicability of TYLCV-Is derived resistance in tomato is discussed.
Annals of Applied Biology 01/2004; 144(1):39 - 44. · 2.18 Impact Factor
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ABSTRACT: Avirus was isolated from Verbena plants that bore virus-like symptoms. The virus, for which the name Verbena latent virus (VeLV) is proposed, was consistently isolated from these plants, both with and without disease symptoms. Electron microscopy studies of ultrathin sections of infected Verbena tissues revealed the presence of elongated flexuous virus particles, ca. 650 nm in length. Its experimental host range was limited to Verbena spp. and Nicotiana clevelandii. No inclusion bodies or specific cytopathological effects, were observed. Electrophoresis of dissociated purified virus preparation in sodium dodecyl sulfate-polyacrylamide gel revealed a major protein component with a molecular mass of 38.9 kDa. Polyclonal antibodies which could specifically bind to virus particles were produced. A portion of the viral RNA was cloned and sequenced; it comprised 2503 nucleotides and contained part of three open reading frames (ORFs) which from the 5' to the 3'-ends, potentially encode for 489 amino acids (ORF1), a 25.8-kDa protein (ORF2) and a 12-kDa protein (ORF3). Comparison of the predicted amino acid sequence with those of other plant viruses revealed 40-60% identity with several carlaviruses. In the light of particle morphology, absence of specific cytopathological effects in ultrathin sections, and genomic and serological properties, it is suggested that this virus belongs to the genus Carlavirus.
Archives of Virology 06/2003; 148(5):1007-15. · 2.11 Impact Factor
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ABSTRACT: Mosaic symptoms were seen on the ornamental plantBupleurum falcatum (Apiaceae) grown in a commercial nursery in Israel. Examination by electron microscopy (EM) of samples from diseased plants
revealed elongated filamentous virus particles. Here we provide evidence thatB. falcatum is an alternate host for Lettuce mosaic potyvirus (LMV). Several lines of evidence indicated thatBupleurum was infected by LMV, including double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), immunosorbent electron
microscopy, and sap and aphid transmission of LMV to lettuce and other host plants. Additionally, general primers derived
from a conserved region of the 3’-terminus of the potyvirus genome were used in a reverse transcription-polymerase chain reaction
(RT-PCR) assay and gave an expected amplification product of 672 bp. A similar approach was carried out to amplify the corresponding
fragment from LMV-infected lettuce. The PCR product was cloned and sequenced; it encompassed an open reading frame encoding
153 amino acids and a 3’ untranslated region (UTR) of 273 nucleotides, very similar to the coat protein (CP) and the 3‵UTR
nucleotide sequence, respectively, of other LMV isolates. Analysis of the amino acid sequences of the putative CP of theBupleurum isolate showed complete identity to the lettuce isolate of LMV. TheBupleurum isolate had no new nucleotide changes which have not been established in other published LMV strain sequences. It is concluded
that LMV is the causal agent of the disease inB. falcatum in Israel.
Phytoparasitica 01/2002; 30(1):88-95. · 0.89 Impact Factor
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ABSTRACT: A method for the differentiation of virus strains based on the shift in electrophoretic mobility of partially annealed RNA transcripts is described. Oppositely oriented RNA transcripts of the NTN- and N-strains of PVY, complementary at their 3'-end variable (strain-specific) region, were annealed to form a partial duplex which moved more slowly in gel than heterologous (NTN+N) unpaired transcripts. Thus, the two virus strains could be identified by annealing to a known reference transcript. The rate of duplex migration was correlated with transcript lengths and could be tightly controlled thereby. Thus, a higher degree of resolution was obtained than with transcript conformation polymorphism, which is empirical and unpredictable in nature.
Journal of Virological Methods 10/2001; 97(1-2):125-31. · 2.01 Impact Factor
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ABSTRACT: An improved procedure for the resolution of RNA transcripts by electrophoretic gel retardation, mediated by annealing to specific homologous oligonucleotiedes is described. The N and NTN strains of PVY served as a model system. Non-polymorphic but sequence-diverse RNA transcripts were copied from PCR products of the two virus strains. The transcripts were resolved by gel electrophoresis, because of the differential retardation effect caused by the binding of strain-specific homologous oligonucleotides. The two PVY strains were thus differentiated. Applicability of this method to virus strain differentiation in general is discussed.
Journal of Virological Methods 04/2001; 92(1):1-4. · 2.01 Impact Factor
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ABSTRACT: ABSTRACT A new virus was isolated from symptomless Aconitum napellus plants. The virus, for which the name Aconitum latent virus (AcLV) is proposed, has flexuous particles 640 nm in length. The experimental host range was limited to Nicotiana clevelandii. Electron microscopy studies of ultrathin sections of infected A. napellus tissues revealed the presence of elongated virus particles. No inclusion bodies characteristic of potyvirus infection were observed. AcLV was purified from naturally infected A. napellus by cesium chloride step gradient centrifugation. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis of dissociated purified virus preparations, a major protein component with a molecular mass of 35 kDa was observed. Diagnostic antibodies that could specifically bind to virus particles were produced. The 5' terminus (620 nucleotides) of the viral RNA was cloned and sequenced. It comprised 71 nucleotides from the untranslated 5' terminus and 549 nucleotides of an open reading frame encoding 183 amino acids. Comparison of the predicted amino acid sequence with those of other plant viruses revealed 40 to 60% identity with several carlaviruses. Based on particle morphology, absence of inclusion bodies in ultrathin sections, the relative molecular weight of the coat protein, the nucleotide sequence, and predicted amino acid homology, it is suggested that this virus belongs to the carlavirus group.
Phytopathology 05/2000; 90(4):340-4. · 2.80 Impact Factor
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ABSTRACT: A method based on differences in electrophoretic mobility of RNA transcripts made from polymerase chain reaction (PCR) products was used for differentiation among virus isolates. A T7 RNA polymerase promoter was attached to amplified prunus necrotic ringspot virus (PNRSV) sequences by PCR. The PCR products then served as a template for transcription. Single-stranded transcripts originated from different PNRSV isolates varied in electrophoretic mobility in polyacrylamide gels, presumably because of transcript conformation polymorphism (TCP). This procedure was applied for the differentiation of PNRSV isolates.
Journal of Virological Methods 10/1998; 74(1):109-15. · 2.01 Impact Factor
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ABSTRACT: The reverse transcriptase-polymerase chain reaction (RT-PCR) was used for detection of prunus necrotic ringspot virus (PNRSV) in dormant peach and almond trees by the application of two different pairs of primers yielding a short and a long product, respectively. The relative amount of the short (200 base pair, bp) product was higher than the longer (785 bp) product. PNRSV was detected better in plant tissues with a low virus concentration (e.g. dormant trees) by amplification of the short PCR product, whereas the long product was product was produced at higher virus titers. Simultaneous amplification of both short and long products was demonstrated using a three-primer mixture in a single reaction tube. In this assay, amplification of either PCR product indicated the presence of PNRSV-specific sequences in the plant tissue examined, thus covering a wide range of virus concentrations in a single test. Dilution of the RNA extracted from infected plant material resulted in a steep decline in the amplification of both short and long PCR products. In contrast, serial dilutions of the intermediate cDNA template differentially affected the amplification patterns: the relative amount of the short product increased whereas that of the long product decreased. These results may explain the preferential amplification of the short PCR product observed in samples containing low virus concentrations.
Journal of Virological Methods 10/1997; 67(2):135-41. · 2.01 Impact Factor
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ABSTRACT: Bean yellow mosaic virus (BYMV) concentration in in-vitro cultured Gladiolus cormlets was low and impossible to detect by the commonly used diagnostic methods. The polymerase chain reaction (PCR) detected viral RNA in most infected cormlets but not in all. Additional amplification of the PCR products by transcription, using T7 RNA polymerase (PCR/T), resulted in virus detection in cases which otherwise went undetected. PCR products having a single polymerase promoter at one end served as a better template for T7 RNA polymerase, and yielded more transcripts of a particular orientation than a template containing promoters at both ends. Repeated cycles of PCR/T resulted in the production of a heterogeneous amplified material which correlated with a progressive decline in amplification rate. Therefore, only the first PCR/T cycle proved to be effective. The PCR/T procedure was shown to be better than other commonly used diagnostic methods including PCR.
Journal of Virological Methods 05/1994; 47(1-2):227-35. · 2.01 Impact Factor
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ABSTRACT: An aphid-transmissible (AT) and two non-aphid-transmissible (NAT) isolates of zucchini yellow mosaic virus (ZYMV) were studied. The predicted amino acid sequences of the coat protein (CP) of the three virus isolates were analysed and compared. The NAT isolates differed from the AT isolate in having a Thr instead of an Ala residue at position 10 in the conserved Asp-Ala-Gly triplet in the N-terminal region of CP. Aphid transmissibility was restored in a progeny virus derived from an infectious clone of the ZYMV-NAT isolate in which Thr was changed back to Ala by site-directed mutagenesis. However this mutation did not have any effect on the multiplication rate in squash, which was significantly higher than that of the AT isolate. The involvement of this mutation in aphid transmission and virus multiplication is discussed.
Journal of General Virology 10/1992; 73 ( Pt 9):2183-7. · 3.36 Impact Factor
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ABSTRACT: A full-length cDNA clone of the RNA genome of the cucurbit potyvirus zucchini yellow mosaic virus (ZYMV) was constructed downstream from a bacteriophage T7 RNA polymerase promoter. A single extra guanosine residue not present in ZYMV RNA was added to the 5' and 3' ends. Capped (m7GpppG) ZYMV RNA transcripts were infectious in 10 of 91 Cucurbita pepo test plants; uncapped RNA transcripts were not infectious. The appearance of symptoms in plants inoculated with the infectious transcript was delayed for more than a week compared to plants inoculated with native viral RNA. The progeny virions recovered from infected plants had the same biological properties (aphid non-transmissibility and typical symptoms) as the parental virus. The progeny virions also reacted positively with ZYMV antiserum and ZYMV-specific probes by dot blot hybridization. The authenticity of the progeny virus was verified by identifying a specific molecular marker (C substituted for T in the 3' non-coding region) using nucleotide sequence analysis.
Journal of General Virology 12/1991; 72 ( Pt 11):2639-43. · 3.36 Impact Factor
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ABSTRACT: Full-length cDNA of genomic RNA of potato virus Y (PVY) was cloned in one piece into a lambda vector. The order of the EcoRI and SalI fragments of the inserted cDNA was determined. This is the first report of the cloning of a long, expressible, potyvirus genome. The availability of such a clone is a prerequisite for any further study of the molecular biology of this group of viruses, as they are expressed into a self-processed primary polyprotein.
Virus Genes 10/1990; 4(3):215-24. · 1.85 Impact Factor
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ABSTRACT: Zucchini yellow mosaic virus (ZYMV) RNA was purified and used as a template for the synthesis of cDNA. A partial restriction map covering 9.4 kb of the ZYMV genome was constructed from three clones designated ZYKS-22, ZYKS-16 and ZYKS-3. Sequencing the 3'-end region of the ZYMV genome indicates the presence of (A)48 chain. This is followed by an untranslated region of 210 nucleotides (nt) and a coding region of 837 nt corresponding to the putative virus coat protein (Cp) gene (cp). The predicted amino acid (aa) sequence of Cp derived from the cDNA showed about 50% to 62% homology with the known aa sequence for Cp of six other potyviruses. A construct of the putative cp was subcloned in frame with the lacZp gene promoter in a Bluescript plasmid and expressed in Escherichia coli cells. The fusion polypeptides (34 and 41 kDa), positively reacted in Western blots with an antiserum prepared against the native virus Cp.
Gene 04/1990; 87(2):273-7. · 2.34 Impact Factor
