Viruses of the genus Allium in the Mediterranean region.

Allium species are economically important crops in the Mediterranean basin. Viruses are among the most important pathogens affecting their yield and especially those belonging to the genera Potyvirus, Carlavirus, and Allexivirus. Members of the genus Potyvirus are usually the most abundant and cause most of the damage induced. Nevertheless, coinfections with different viruses are not scarce, especially in garlic, and can have synergistic effects that lead to even greater crop losses. Vegetative propagation of alliums and the transmission of most of their viruses by arthropod vectors have significantly contributed to their wide dissemination in the Mediterranean region and elsewhere in the world. Here, we review the general biological and molecular features, the epidemiology, incidence, and methods of diagnosis of the most widespread allium viruses in the basin. Control measures are proposed depending on the mode of propagation of the various alliums, the epidemiology of their viruses and the cultivation procedures adapted by the Mediterranean farmers. The importance of the production and use of virus-free propagative material in order to combat viral diseases of allium crops is especially highlighted. A final discussion focuses on the main shortages identified in the research area of allium viruses, and proposals are made for putative future developments.

Molecular characterization of allium virus X, a new potexvirus in the family Alphaflexiviridae, infecting ornamental allium.

ABTRACT: A new potexvirus affecting ornamental allium spp in the Netherlands was identified and characterized at the molecular level. The virus had a single-stranded RNA genome of 7100 bp (excluding the 18 bp poly A tail). The genome organization was found to be typical of members of the genus Potexvirus and consisted of five open reading frames (ORF). Nucleotide and amino acid sequence comparisons with those of known potexvirus members showed that this virus is related to Hosta virus X and Hydrangea ringspot virus. Sequence similarities and phylogenetic relationships suggested that the allium virus is a new and distinct species in the genus Potexvirus and the name, Allium virus X (AlVX) is proposed.

Cryopreservation of garlic germplasm collections using the droplet-vitrification technique.

The droplet-vitrification protocol was applied to unripe inflorescences of plants of two Korean garlic collections, Danyang and Mokpo, to establish a cryopreserved germplasm collection. Garlic unripe inflorescences of the 59 accessions harvested at Danyang showed a mean survival of 83.3% and regeneration of 73.5% after cryopreservation. Unripe inflorescences of accessions cryopreserved at sub-optimal developmental stages displayed lower survival and/or regeneration. Of these 59 accessions, 53 were cryopreserved and stored for long-term conservation. In the Mokpo collection, unripe inflorescences of 149 accessions were cryopreserved, displaying a mean survival of 79.9% and regeneration of 78.2%. Of these 149 accessions, 116 were cryopreserved and stored for the long-term. A total of 252 accessions of five clonal Allium species, including garlic, were cryopreserved using unripe inflorescences, cloves or bulbils, with a mean survival of 80.9% survival and regeneration of 77.0%, from which 221 accessions were stored in liquid nitrogen for long-term conservation. The real-time quantitative, reverse transcription (RT)-PCR assay of several garlic viruses showed that virus concentration was much lower in plantlets originating from cryopreserved material, compared to plantlets originating from preculture control and dehydration control samples. These results demonstrate that large-scale implementation of cryopreservation of Allium germplasm is feasible and that it can result in the regeneration of virus-free or little infected material. These findings will strongly facilitate the conservation and international exchange of Allium germplasm.

Characterisation of the welsh onion isolate of Shallot yellow stripe virus from China.

The host range and nucleotide sequence of shallot yellow stripe virus (SYSV) from welsh onion in Shandong province, China is described. Of the plants tested, only shallot and welsh onion became infected but most shallot plants were symptomless. The complete sequence of one isolate (10429 nt) and the 3'-terminal 3540 nts of a second isolate were determined. They had c. 90% nt identity to one another and to published (partial) sequences of SYSV. SYSV was most closely related to onion yellow dwarf virus (OYDV) and resembled it in having a much larger P3 protein than other species in the genus.

Characterisation of a potyvirus and a potexvirus from Chinese scallion.

Molecular analyses of viruses infecting Chinese scallion (Allium chinense G. Don) showed that the plants did not contain any of the poty-, carla- or allexiviruses that are common in garlic plants in China. The complete sequences of a potyvirus and a potexvirus were determined and these were shown to represent different viruses from any in the databases. They could be transmitted mechanically to scallion but not to other Allium species (including garlic) or to Narcissus. The potyvirus, tentatively named Scallion mosaic virus, has a distant relationship (c. 62% nucleotide identity over the entire genome) to Turnip mosaic virus and Japanese yam mosaic virus, with which it grouped in phylogenetic analyses. Its genome is 9324 nts long, encoding a 341.3 kDa polyprotein of 3001 amino acids. The potexvirus, tentatively named Scallion virus X, has a genome 6987 nts long and its organisation was similar to that of the other potexviruses but with only 46.3-63.2% nucleotides identical to them. It is most closely related to Narcissus mosaic virus but phylogenetic analyses indicate that it should be considered a distinct species. Neither of the viruses have been detected in garlic, although the two host plants are closely related.

Characterisation of some carla- and potyviruses from bulb crops in China. Brief report.

Conserved carla- and potyvirus primers were used in RT-PCR to amplify virus fragments from garlic and other bulb crops in China and the fragments were subsequently sequenced and compared in phylogenetic analyses. Garlic plants from Henan, Hubei, Jiangsu, Shangdong and Yunnan provinces all contained at least one isolate each of Garlic latent virus (genus Carlavirus), Onion yellow dwarf virus (OYDV, genus Potyvirus) and Leek yellow stripe virus (LYSV, genus Potyvirus). The complete sequence of a Zhejiang isolate of LYSV was also determined, providing the first complete sequence of this virus. The genome was 10142 nucleotides long excluding the poly(A) tail and had the typical features of the genus Potyvirus, although some of the amino acids surrounding the polyprotein cleavage sites were unusual. Shallot yellow stripe virus (SYSV) was amplified from bunching onion (Allium fistulosum var. caespitosum) in Zhejiang province, providing the first record of SYSV in China. Lily mottle virus was amplified from dragon-teeth lily (Lilium brownii var. viridulum).

Detection of infectious viral particles in plant protoplasts inoculated with transcripts of full-length shallot virus X cDNA.

Flexible filamentous shallot virus X (ShVX) particles were detected in extracts of Beta vulgaris protoplasts inoculated with transcripts from a full-length ShVX cDNA. Extracts from ShVX-infected protoplast were infectious for ShVX-healthy shallot seedlings. Western blot analysis of inoculated plants revealed the accumulation of the ShVX coat protein, while electron microscopy confirmed the presence of ShVX virions. The results suggest that the in vitro RNA transcripts from full-length ShVX cDNA give rise to infectious viral particles.

Differentiation of Allium carlaviruses isolated from different parts of the world based on the viral coat protein sequence.

Common primers which amplify the 3' terminal genomic RNAs of Allium carlaviruses were designed based on the nucleotide sequence of shallot latent virus (SLV), garlic latent virus (GLV) and garlic common latent virus (GCLV). A total of fifteen cDNAs encoding the coat protein (CP) of the carlaviruses, including the biologically identified isolates SLV, GLV and GCLV as well as viruses from infected Allium plants cultivated in different parts of the world, were amplified by RT-PCR with the common primers. The cDNAs were then cloned and sequenced. The predicted viral CP amino acid sequence as well as the nucleotide sequence revealed that SLV and GLV, previously considered as separate viruses on the basis of their biological and physical properties, belong to the same species of the genus Carlavirus. Both viruses are clearly differentiated from GCLV. In addition, every SLV and GLV isolate from the Allium plants in Taiwan showed characteristic and common variations in their CP sequences, suggesting the possible presence of geographical variants. However, no apparent sequence variations of SLV and GLV related to their host plant species, including A. sativum, A. wakegi, A. chinense, A. fistulosum, A. cepa and A. ampeloprasum, were observed. These findings suggested that the sequence variations observed in the respective virus isolates do not correlate with the specificity of their infectivities for Allium species.

Nucleotide sequence analysis of virus isolates indicates the presence of three potyvirus species in Allium plants.

cDNAs of potyviruses from Allium plants cultivated in different parts of the world were cloned by RT-PCR with a common primer for amplifying the 3' terminal genomic RNAs of onion yellow dwarf virus (OYDV), leek yellow stripe virus (LYSV) and, probably, of closely related potyviruses. Their nucleotide sequences bearing the viral coat protein (CP) gene and the 3' non-coding sequence were determined and compared. The degree of their sequence similarities clearly differentiated the respective viruses into 3 groups, namely OYDV "garlic-type", "wakegi-type" and LYSV group. The "garlic-type" included all the garlic isolates and two Indonesian shallot isolates. The "wakegi-type" group consisted of the isolates from Indonesian shallot and previously reported ones from Japanese Allium plants excluding garlic. The LYSV group was represented by LYSV isolates from garlic and leek. The CP sequences of LYSV group viruses differed from those of OYDV "garlic-type" and "wakegi-type" viruses (less than 60% similarities). In contrast, the sequence similarities between the OYDV "wakegi-type" and "garlic-type" isolates were 73.5 to 76.7%, suggesting they were closely related but should be discriminated as distinct species. These findings indicate that at least three distinct potyviruses, clearly distinguishable by the viral CP sequence, are present in Allium species. Finally, we concluded that the "garlic-type" viruses correspond to the typical OYDV and the "wakegi-type" viruses represent the viruses previously identified as Welsh onion yellow stripe virus (WoYSV) and shallot yellow stripe virus (SYSV). We propose the name wakegi yellow dwarf virus (WYDV) for the "wakegi-type" viruses.

Nucleotide sequences of the 3' terminal region of onion yellow dwarf virus isolates from Allium plants in Japan.

The 2032 nucleotide sequence of the 3' terminal region of onion yellow dwarf virus (OYDV) isolated from Allium wakegi, bearing the genes for viral coat protein (CP) and a truncated RNA-dependent RNA polymerase, has been determined. Respective homologies of the nucleotide sequence in the corresponding region and the deduced amino acid sequence of CP with the equivalents of leek yellow stripe virus (LYSV) from garlic were 68.0 and 59.3%. Variation in the nucleotide sequence is concentrated in the boundary region between the putative RNA-dependent RNA polymerase gene and the CP gene as well as in the 3' noncoding region. These sequence divergencies, including the deletion of 79 nucleotides, resulted both in alterations to the amino acid sequence and the absence of 28 amino acid residues in the amino terminal region of OYDV CP in comparison with LYSV CP. In addition, the length of the 3' noncoding sequence of OYDV was one-third that of LYSV. Comparison of the 3' terminal 1197 nucleotides sequence of OYDV with sequences of the respective cDNAs cloned by RT-PCR directly from the total RNA of infected Allium plants that included two varieties of A. fistulosum, "Wakenegi" and "Shimonita-negi", and A. chinense, showed 90.7% overall identities, even though they have long been cultivated in locally restricted area in Japan. These findings appear to suggest that a single strain of OYDV invaded Japanese Allium plants long ago and spread throughout them.

[42K protein of shallot X virus is expressed in infected Allium species plants]. / 42K-belok X-virusa shalota ékspressiruetsia v infitsirovannykh roda Allium.

The main difference between genome structures of shallot virus X (ShVX) and related potex- and carlaviruses is the unique gene of ShVX coding for a 42K protein. The amino acid sequence of this protein was analyzed and compared with those of similar proteins from several newly characterised viruses of garlic. Using antibodies against the recombinant 42K protein, expression of the 42K protein of ShVX was detected in most of plants where the ShVX coat protein is present.