Grapevine Chrome Mosaic Virus Research Articles

Grapevine chrome mosaic virus (chrome mosaic of grapevine)

Grapevine chrome mosaic virus (chrome mosaic of grapevine)

Next generation sequencing and molecular analysis of artichoke Italian latent virus.

Next-generation sequencing (NGS) allowed the assembly of the complete RNA-1 and RNA-2 sequences of a grapevine isolate of artichoke Italian latent virus (AILV). RNA-1 and RNA-2 are 7,338 and 4,630 nucleotides in length excluding the 3' terminal poly(A) tail, and encode two putative polyproteins of 255.8kDa (p1) and 149.6kDa (p2), respectively. All conserved motifs and predicted cleavage sites, typical for nepovirus polyproteins, were found in p1 and p2. AILV p1 and p2 share high amino acid identity with their homologues in beet ringspot virus (p1, 81% and p2, 71%), tomato black ring virus (p1, 79% and p2, 63%), grapevine Anatolian ringspot virus (p1, 65% and p2, 63%), and grapevine chrome mosaic virus (p1, 60% and p2, 54%), and to a lesser extent with other grapevine nepoviruses of subgroup A and C. Phylogenetic and sequence analyses, all confirmed the strict relationship of AILV with members classified in subgroup B of genus Nepovirus.

The sequencing of the complete genome of a Tomato black ring virus (TBRV) and of the RNA2 of three Grapevine chrome mosaic virus (GCMV) isolates from grapevine reveals the possible recombinant origin of GCMV.

The complete genome of a Tomato black ring virus isolate (TBRV-Mirs) (RNA1, 7,366 nt and RNA2, 4,640 nt) and the RNA2 sequences (4,437; 4,445; and 4,442 nts) of three Grapevine chrome mosaic virus isolates (GCMV-H6, -H15, and -H27) were determined. All RNAs contained a single open reading frame encoding polyproteins of 254 kDa (p1) and 149 kDa (p2) for TBRV-Mirs RNA1 and RNA2, respectively, and 146 kDa for GCMV RNA2. p1 of TBRV-Mirs showed the highest identity with TBRV-MJ (94 %), Beet ringspot virus (BRSV, 82 %), and Grapevine Anatolian ringspot virus (GARSV, 66 %), while p2 showed the highest identity with TBRV isolates MJ (89 %) and ED (85 %), followed by BRSV (65 %), GCMV (58 %), and GARSV (57 %). The amino acid identity of RNA2 sequences of four GCMV isolates (three from this study and one from GenBank) ranged from 91 to 98 %, the homing protein being the most variable. The RDP3 program predicted putative intra-species recombination events for GCMV-H6 and recognized GCMV as a putative inter-species recombinant between GARSV and TBRV. In both cases, the recombination events were at the movement protein level.

Magyarországon előforduló szőlővírusok és a hazai szőlő vírusbetegségek áttekintése

The past fifty years important advances have been made in the field of grapevine virus research, including characterization of pathogens and control measurements. Still the occurrence of Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Tomato black ring virus (TBRV), Grapevine chrome mosaic virus (GCMV), Alfalfa mosaic virus (AMV), Grapevine Bulgarian latent virus (GBLV), Grapevine fleck virus (GFkV), Grapevine leafroll- associated viruses (GLRaV1-4), Grapevine virus A (GVA), Grapevine virus B (GVB) and Grapevine rupestris stem pitting- associated virus (GRSPaV) have been reported in Hungary and characterized by conventional methods as woody indexing, herbaceous indexing and serological methods. Among grapevine viruses the Grapevine line pattern virus (GLPV) seems to be uncial; because it was reported only in Hungary. Causal agents of several grapevine diseases, like enation, vein necrosis and vein mosaic remained undiscovered. These virus-like diseases occurred only sporadically, without economic importance.

Detecção e identificação molecular de vírus associados a videiras sintomáticas e assintomáticas

A propagação vegetativa da videira favorece infecções virais múltiplas, com expressão diferencial de sintomas em função da combinação da cultivar ou espécie da hospedeira com a espécie viral. Os objetivos deste trabalho foram detectar e identificar as espécies virais presentes em duas espécies/cultivares de videira: uma sintomática e outra assintomática. DsRNA de ambas as amostras foi submetido à RT-PCR com 17 pares de oligonucleotídeos específicos para a detecção de Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine virus D (GVD), Grapevine fleck virus (GFkV), Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Grapevine chrome mosaic virus (GCMV), Rupestris stem pitting-associated virus (RSPaV), Grapevine leafroll-associated virus 1 a 4 (GLRaV-1 a -4), além de três pares de oligonucleotídeos degenerados. Pelo menos um fragmento amplificado, por par de oligonucleotídeos, foi clonado e sequenciado. Plantas sintomáticas e assintomáticas mostraram infecções múltiplas por RSPaV, GLRaV-2 e/ou GLRaV-3. As sequências de nucleotídeos obtidas para sete isolados de RSPaV, três de GLRaV-2 e dois de GLRaV-3 apresentaram identidades superiores a 90% com espécies homólogas e permitiram a definição das possíveis estirpes presentes nas amostras infectadas. Esses resultados evidenciam a necessidade da diagnose viral baseada em testes específicos para determinar a condição sanitária da videira.

Pathogen‐tested material of grapevine varieties and rootstocks

Pathogen‐tested material of grapevine varieties and rootstocks

Development of degenerate and species-specific primers for the differential and simultaneous RT-PCR detection of grapevine-infecting nepoviruses of subgroups A, B and C

Based on the nucleotide sequence homology of RNA-1 and RNA-2 of nepoviruses isolated from grapevines, three sets of degenerate primers, one for each of the three subgroups of the genus (A, B and C), were designed and proved effective for RT-PCR detection of subgroups in infected grapevines and herbaceous hosts. Primers designed specifically for detecting subgroup A species amplified a fragment of 255 bp from samples infected by Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Tobacco ringspot virus (TRSV) and Grapevine deformation virus (GDefV), but not from samples infected by other nepovirus species. Similarly, primers for detection of subgroup B nepoviruses amplified a 390 bp product from samples infected by Grapevine chrome mosaic virus (GCMV), Tomato black ring virus (TBRV), Grapevine Anatolian ringspot virus (GARSV) and Artichoke Italian latent virus (AILV). The third set of primers amplified a 640 bp fragment, only from samples infected by subgroup C nepoviruses, i.e Tomato ringspot virus (ToRSV) Grapevine Bulgarian latent virus (GBLV), and Grapevine Tunisian ringspot virus (GTRSV). These primers were able to detect simultaneously all viral species belonging to the same subgroup and to discriminate species of different subgroups. Multiplex-PCR detection of subgroup A and B nepoviruses was obtained using a specific primer ( sense for subgroup A and antisense for subgroup B) for each of the species of the same subgroup in combination with the degenerate subgroup-specific primers. In this way it was possible to detect four different viral species in single samples containing mixtures of viruses of the same subgroup. In particular, for viruses of subgroup A (TRSV, GFLV, ArMV and GDefV) amplicons of 190, 259, 301 and 371 bp were obtained, whereas amplicons of 190, 278, 425 and 485 bp, respectively, were obtained from samples infected with viruses of subgroup B (GCMV, AILV, GARSV and TBRV).

Complete Nucleotide Sequence of the RNA-2 of Grapevine Deformation and Grapevine Anatolian Ringspot Viruses

The nucleotide sequence of RNA-2 of Grapevine Anatolian ringspot virus (GARSV) and Grapevine deformation virus (GDefV), two recently described nepoviruses, has been determined. These RNAs are 3753 nt (GDefV) and 4607 nt (GARSV) in size and contain a single open reading frame encoding a polyprotein of 122 kDa (GDefV) and 150 kDa (GARSV). Full-length nucleotide sequence comparison disclosed 71-73% homology between GDefV RNA-2 and that of Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV), and 62-64% homology between GARSV RNA-2 and that of Grapevine chrome mosaic virus (GCMV) and Tomato black ring virus (TBRV). As previously observed in other nepoviruses, the 5' non-coding regions of both RNAs are capable of forming stem-loop structures. Phylogenetic analysis of the three proteins encoded by RNA-2 (i.e. protein 2A, movement protein and coat protein) confirmed that GDefV and GARSV are distinct viruses which can be assigned as definitive species in subgroup A and subgroup B of the genus Nepovirus, respectively.

New Australian record for Xiphinema vuittenezi on Vitis vinifera

Xiphinema vuittenezi is reported for the first time in Australia in soil associated with roots of declining grapevines (Vitis vinifera) in a Barossa Valley (SA) vineyard. This nematode may be able to transmit some strains of Cherry leaf roll virus and is the putative vector of Grapevine chrome mosaic virus; hence its finding may have potential significance for the spread of some nepoviruses in grapes and other hosts.

PROPERTIES OF A PREVIOUSLY UNDESCRIBED NEPOVIRUS FROM SOUTH-EAST ANATOLIA

SUMMARY A virus with isometric particles ca. 30 nm in diameter and angular contour was isolated by inoculation of sap from a Turkish grapevine showing very mild fanleaf symptoms. In sucrose density gradient centrifugation the virus sedimented as three components: T (empty shells), M (particles containing a RNA species with mol. wt 1.4 x 10 6 Da) and B (particles containing a RNA species with mol. wt 2.2 x 10 6 Da). The coat protein subunits were of a single type with Mr ca. 55.5 kDa. An antiserum with a titre of 1:256 was raised, which did not react with healthy plant antigens. A fragment 1,274 nt in size, representing the 3’ terminal region of genomic RNA-2 was sequenced. Direct comparison analysis with sequences from the same region from GenBank-EMBL databases revealed variable levels of homology with other grapevine nepoviruses, the closest being Grapevine chrome mosaic virus (GCMV) (62% identity at the amino acid level), Artichoke Italian latent virus (AILV) and Tomato blackring virus (TBRV) (49% identity at the amino acid level with both). Based on the determined sequence, specific primers were designed which in RTPCR assays amplified a 343 bp fragment virus from grapevine crude tissue extracts. Infected N. occidentalis leaves had a cytopathology comparable to that of nepovirus infections, typical virus-containing tubules, mostly associated with plasmodesmata. The physicochemical and ultrastructural properties of this virus resembled very much those of nepoviruses. However, it was serologically unrelated to 17 different members of this genus, including all those recorded from grapevines. These results support the notion that the Turkish grapevine virus is a hitherto undescribed member of the genus Nepovirus belonging in subgroup B, for which the name Grapevine Anatolian ringspot virus (GARSV) is proposed.

Multiplex Polymerase Chain Reaction Identification of Single Individuals of the Longidorid Nematodes Xiphinema index, X. diversicaudatum, X. vuittenezi, and X. italiae Using Specific Primers from Ribosomal Genes

ABSTRACT The species X. index, X. diversicaudatum, X. vuittenezi, and X. italiae are established (E) or putative (P) vectors of Grapevine fanleaf virus (GFLV) (E), Arabis mosaic virus (E), Grapevine chrome mosaic virus (P), and GFLV (P) nepoviruses of grapevine, respectively. All four species are very closely related taxonomically and their low field densities make them difficult to identify from morphological and morphometrical diagnostic characters when only single or few individuals are detected. To improve diagnostic accuracy, a simple method was developed. The internal transcribed spacer 1 (ITS1) region spanning the 18S and 5.8S ribosomal genes was sequenced in one population of each species using two conserved primers from these genes. The ITS1 fragments were 1,132 bp (X. vuittenezi), 1,153 bp (X. index), 1,175 bp (X. diversicaudatum), and 1,190 bp (X. italiae), i.e., a difference of over 5% between the extremes. The sequence variability made it possible to design species-specific internal sense primers that amplified, in combination with the same antisense ITS1 primer, a single signature fragment (340 bp for X. index, 414 bp for X. italiae, 591 bp for X. vuittenezi, and 813 bp for X. diversicaudatum). Tests with DNA from a single adult or juvenile nematode confirmed the specificity of the primers from diverse isolates or populations. The primers were successfully used in a multiplex test for the reliable detection of two to four mixed species, each represented by a single individual. This multiplex-based diagnostic tool will be particularly useful for successful nematode management practices in vineyards.

Nucleotide sequence and taxonomy of Cycas necrotic stunt virus. Brief report.

Cycas necrotic stunt virus (CNSV) is the only well-characterized virus from gymnosperm. cDNA segments corresponding to the bipartite genome RNAs (RNA1, RNA2) were synthesized and sequenced. Each RNA encoded a single polyprotein, flanked by the 5' and 3' non-coding regions (NCR) and followed by a poly (A) tail. The putative polyproteins encoded by RNA1 and RNA2 had sets of motifs, which were characteristic of viruses in the genus Nepovirus. The polyproteins showed higher sequence identities to Artichoke Italian latent virus, Grapevine chrome mosaic virus and Tomato black ring virus, all of which belong to subgroup b of the genus Nepovirus, than to other nepoviruses. Phylogenetic analysis of RNA dependent RNA polymerase and coat protein also showed closer relationships with these viruses than other viruses. The data obtained supported the taxonomical status of CNSV as a definitive member of the genus Nepovirus, subgroup b.

Agrobacterium rhizogenes and A. tumefaciens co-transformation to obtain grapevine hairy roots producing the coat protein of grapevine chrome mosaic nepovirus

Hairy root cultures of grapevine were obtained from plantlets co-inoculated by virulent Agrobacterium rhizogenes strains and disarmed A. tumefaciens strains harbouring the binary vectors pKHG4 and pKVHG 2+. These plasmids contain the nptII, hpt and gus genes and differ for the presence of the gene encoding for the grapevine chrome mosaic virus coat protein. For the cultivar ‘Gravesac’, 72% of the excised root tips initiated hairy root cultures on growth regulator-free media. According to the nature of the strains used in co-inoculation, co-transformation frequencies of the hairy root clones ranged from 4 to 16%. Co-transformed roots showed resistance to kanamycin and hygromycin but responses varied from clone to clone. Fluorometric GUS expression and GCMV coat protein production showed a large variability among hairy root clones co-transformed by pKHVG2+. Though the presence of gus, nptII and GCMV coat protein genes was checked by polymerase chain reaction and Southern blotting, it was difficult to establish a clear relationship between expression of the different transgenes. The regeneration of plants was not achieved, but the possibility to graft in vitro transgenic roots to non transformed shoot systems could permit rapid testing of the resistance induced by nepovirus coat protein in roots of cultivars that are recalcitrant to A. tumefaciens-mediated transformation.

Specificity in the Association of Tomato Black Ring Virus Satellite RNA with Helper Virus

The satellite RNAs (sat-RNAs) associated with some isolates of tomato black ring virus (TBRV) consist of single-stranded molecules of about 1375 nucleotides, encoding a nonstructural protein of 48K which has been shown to be involved in the replication of the sat-RNA. The TBRV sat-RNAs are also dependent for their replication and for their encapsidation on the helper virus. To characterize the nature of the association between sat-RNA and helper virus, transcripts of sat-RNA from TBRV isolates C and L (respectively, of serotypes G and S) have been prepared and inoculated onto Chenopodium quinoa leaves or protoplasts. Transcript of the TBRV sat-RNA C is efficiently multiplied when coinoculated with the genomic RNAs of TBRV isolate G (used instead of TBRV isolate C, because isolate G was depleted of sat-RNA), but does not multiply with TBRV isolate L. On the other hand, transcript of the sat-RNA L is able to multiply with the cognate helper virus and, less efficiently, with grapevine chrome mosaic virus (another nepovirus, 80% similar to TBRV), but does not multiply with TBRV G. The specificity of the association resides at the level of sat-RNA replication. Analysis of the multiplication of chimeric sat-RNAs, obtained by exchanging different regions between the two sat-RNAs C and L, showed that the 5′ and the 3′ noncoding regions of the sat-RNA, although important for replication, are not implicated in specificity. The results suggest that the determinants of the specificity are contained in the 48K sat-RNA-encoded protein.

Transfer of the 3' non-translated region of grapevine chrome mosaic virus RNA-1 by recombination to tomato black ring virus RNA-2 in pseudorecombinant isolates.

In grapevine chrome mosaic and tomato black ring viruses (GCMV and TBRV), as in many other nepoviruses, the 3' non-translated regions (3'NTR) are identical between the two genomic RNAs. We have investigated the structure of the 3'NTR of two recombinant isolates which contain GCMV RNA-1 and TBRV RNA-2. In these isolates, the 3'NTR of RNA-1 was transferred to RNA-2, thus restoring the 3' identity. The transfer occurred within three passages, and probably contributes to the spread of randomly appearing mutations from one genomic RNA to the other. The site of recombination is near the 3' end of the open reading frame.

Amino-acid sequence comparison of nepovirus coat proteins.

The amino acid sequence of the coat proteins of several nepoviruses was determined by a combination of peptide and nucleic acid sequencing (grapevine fanleaf virus, arabis mosaic virus, tomato blackring virus, grapevine chrome mosaic virus). These sequences were compared and showed homologies ranging from 10% to 69%, and 96.7% for the two arabis mosaic virus strains. 10% homology does not reflect any relationship between viruses, and our results implicate, that nepoviruses, considering the homology of the coat protein sequences of viruses as a parameter for virus taxonomy, may be divided into several subgroups.

In vitro processing of the RNA-2-encoded polyprotein of two nepoviruses: tomato black ring virus and grapevine chrome mosaic virus

In vitro translation of RNA-2 of each of two closely related nepoviruses, tomato black ring virus (TBRV) and grapevine chrome mosaic virus (GCMV), in a rabbit reticulocyte lysate resulted in the synthesis of single polypeptides of 150K and 146K respectively. Processing of these polyproteins occurred after the addition of translation products of homologous RNA-1. The positions of the cleavage products within the polyproteins were determined. From the N to the C terminus, Mr values for the proteins were 50K, 46K and 59K for TBRV and 44K, 46K and 56K for GCMV. TBRV RNA-1 translation products also cleaved the polyproteins encoded by GCMV RNA-2 which suggests that the cleavage sites in the two polyproteins are similar.

RNA2 of grapevine fanleaf virus: sequence analysis and coat protein cistron location

The nucleotide sequence of the genomic RNA2 (3774 nucleotides) of grapevine fanleaf virus strain F13 was determined from overlapping cDNA clones and its genetic organization was deduced. Two rapid and efficient methods were used for cDNA cloning of the 5' region of RNA2. The complete sequence contained only one long open reading frame of 3555 nucleotides (1184 codons, 131K product). The analysis of the N-terminal sequence of purified coat protein (CP) and identification of its C-terminal residue have allowed the CP cistron to be precisely positioned within the polyprotein. The CP produced by proteolytic cleavage at the Arg/Gly site between residues 680 and 681 contains 504 amino acids (Mr 56019) and has hydrophobic properties. The Arg/Gly cleavage site deduced by N-terminal amino acid sequence analysis is the first for a nepovirus coat protein and for plant viruses expressing their genomic RNAs by polyprotein synthesis. Comparison of GFLV RNA2 with M RNA of cowpea mosaic comovirus and with RNA2 of two closely related nepoviruses, tomato black ring virus and Hungarian grapevine chrome mosaic virus, showed strong similarities among the 3' non-coding regions but less similarity among the 5' end non-coding sequences than reported among other nepovirus RNAs.

Cloning full-length cDNA of grapevine chrome mosaic nepovirus

Full-length cDNA copies of the genomic RNAs of grapevine chrome mosaic virus were obtained and cloned in Escherichia coli by a one-step procedure. The cloning protocol included size selections by agarose-gel electrophoresis of both the single-stranded and the double-stranded full-length cDNAs. First-strand cDNA synthesis was primed with oligodeoxythymidine while second-strand synthesis was primed with specific synthetic oligodeoxynucleotides, allowing cloning of the 3′ poly(A) and of the last 5′ nucleotides of the viral RNA template. For the 7.2-kb and 4.4-kb viral RNAs, up to 20% and 80%, respectively, of the clones were found to be full-length. Even for large templates, this procedure allows fast and efficient cloning of full-length cDNAs.