Molecular characterisation of Onion yellow dwarf virus (OYDV) infecting garlic (Allium sativum L.) in Israel: Thermotherapy inhibits virus elimination by meristem tip culture

Garlic (cv. Shani) was tested using single step RT‐PCR and digoxygenin (DIG) labelled dot‐blot for a number of viruses. Following sequence analysis it was shown that at least three different polymorphs of the potyvirus Onion yellow dwarf virus (OYDV) infect the same plant simultaneously, together with the potyvirus Leek yellow stripe virus (LYSV), the carlavirus Garlic common latent virus (GCLV) and a multitude of allexiviruses (Shallot virus X (ShVX) related viruses]. Several garlic plants free of all the viruses tested were obtained through meristem‐tip culture. Plants infected with single viruses or with different combinations of viruses were similarly obtained. Meristem‐tip culture was confirmed as a satisfactory method of virus eradication, while thermotherapy treatment given to mother plantlets before meristem excision was found to specifically antagonise OYDV eradication. This work uses molecular methods for the first time to examine the effectiveness of meristem‐tip culture for the eradication of multiple viruses from garlic.     

Detection of Garlic Viruses Using SYBR Green Real‐time Reverse Transcription‐Polymerase Chain Reaction

SYBR Green real‐time RT‐PCR assay was developed and optimized for the sensitive detection of Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Garlic common latent virus (GCLV), Shallot latent virus (SLV) and Mite‐borne filamentous virus (MbFV). The polyvalence of the designed primers was tested on 50 genotypes of garlic (Allium sativum L.) which originated from different countries. Plasmid standards were prepared and used as positive standards. The efficiencies of all reactions were 97, 93, 99, 98 and 87% for OYDV, LYSV, SLV, GCLV and MbFV standards, respectively. The detection limit for OYDV, LYSV and GCLV was as low as five gene copies, for SLV it was 15 gene copies and for MbFV it was 130 gene copies. In comparison with ELISA, more virus‐positive garlic accessions were detected with LYSV and GCLV by SYBR Green‐based real‐time RT‐PCR assay. This method was shown to be a more suitable tool for the detection of highly variable pathogens, such as garlic viruses.     

Fluctuations in concentration of two potyviruses in garlic during the growing period and sampling conditions for reliable detection by ELISA

To optimise sampling conditions for the detection by ELISA of Onion yellow dwarf virus (OYDV) and Leek yellow stripe virus (LYSV), the most important viral pathogens of garlic worldwide, relative virus concentrations were determined during the growing period and in different leaf parts by DAS‐ELISA. Both viruses were found to have uneven distributions in garlic plants, with the tips of the two latest fully developed leaves showing the highest concentrations and the oldest leaves the lowest concentrations. The tips of the youngest leaves were found to have higher virus concentrations than their middle and basal sections. In the older leaves, viruses were distributed more uniformly in the three leaf sections. In the oldest leaves virus levels in the leaf tips were significantly decreased. The concentrations of OYDV and LYSV increased until March, whereas later on they decreased. During storage of leaf samples at 6°C for 15 days, a loss was found of both virus antigens of more than 80%, and during 109 days of storage at ?30°C a loss of more than 90% was found.     

The Global Trade in Fresh Produce and the Vagility of Plant Viruses: A Case Study in Garlic

As cuisine becomes globalized, large volumes of fresh produce are traded internationally. The potential exists for pathogens infecting fresh produce to hitchhike to new locations and perhaps to establish there. It is difficult to identify them using traditional methods if pathogens are novel, scarce, and/or unexpected. In an attempt to overcome this limitation, we used high-throughput sequencing technology as a means of detecting all RNA viruses infecting garlic (Allium sativum L.) bulbs imported into Australia from China, the USA, Mexico, Argentina and Spain, and those growing in Australia. Bulbs tested were grown over multiple vegetative generations and all were stably infected with one or more viruses, including two species not previously recorded in Australia. Present in various combinations from 10 garlic bulbs were 41 virus isolates representing potyviruses (Onion yellow dwarf virus, Leek yellow stripe virus), carlaviruses (Shallot latent virus, Garlic common latent virus) and allexiviruses (Garlic virus A, B, C, D, and X), for which 19 complete and 22 partial genome sequences were obtained, including the first complete genome sequences of two isolates of GarVD. The most genetically distinct isolates of GarVA and GarVX described so far were identified from Mexico and Argentina, and possible scenarios explaining this are presented. The complete genome sequence of an isolate of the potexvirus Asparagus virus 3 (AV3) was obtained in Australia from wild garlic (A. vineale L.), a naturalized weed. This is first time AV3 has been identified from wild garlic and the first time it has been identified beyond China and Japan. The need for routine generic diagnosis and appropriate legislation to address the risks to primary production and wild plant communities from pathogens spread through the international trade in fresh produce is discussed.     

The Distribution of Garlic Viruses in Leaves and Bulbs during the First Year of Infection

Garlic plants are naturally infected with a mixture of viruses. Virus‐free garlic plants, obtained by meristem culture, rapidly become reinfected when planted in the field. With the aim of understanding virus movement and fluctuations in virus concentration in leaves and cloves of garlic plants in the first year after infection, Onion yellow dwarf virus, Leek yellow stripe virus, and other viruses were analyzed by double‐antibody sandwich enzyme‐linked immunosorbent assay. Significant differences were detected in virus concentration in different leaves, but the distribution of the viruses was variable. Therefore, no one type or position of leaf is preferable for detecting virus presence. Instead, sampling any leaf at the end of the crop cycle, about 200 days after planting, is advisable because virus concentration is several times higher in older plants. The analysis of virus distribution in bulbs revealed that virus concentration was higher in early‐inoculated than in late‐inoculated plants. In 81% of the bulbs, cloves were either all positive or all negative in serological tests. Only in 6% of the cases were positive and negative cloves found in the same bulb, and in 13% of the bulbs, negative results coexisted with an uncertain status. The tests of virus concentration in relation to the layers of each bulb revealed important differences. Only the innermost layer showed differences with other layers, but this was poorly represented as it had fewer cloves.     

Serological Relationships Among the Over-expressed Coat Proteins of Allexiviruses

Coat protein genes of six members of the genus Allexivirus, Garlic viruses ‐A, ‐B, ‐C, ‐D, ‐E and ‐X, were over‐expressed in bacteria and used to prepare polyclonal antibodies. In western blot analysis, Garlic virus D (GarV‐D) and GarV‐E were closely related serologically and there were weaker relationships between some other pairs, particularly GarV‐E with GarV‐A, GarV‐X with GarV‐A, GarV‐X with GarV‐B and GarV‐X with GarV‐C. Sequence analysis suggested that there was no simple relationship between amino acid identity and serological properties.     

The ever increasing diversity of begomoviruses infecting non‐cultivated hosts: new species from Sida spp. and Leonurus sibiricus,plus two New World alphasatellites

Begomoviruses (whitefly‐transmitted, single‐stranded DNA plant viruses) are among the most damaging pathogens causing epidemics in economically important crops worldwide. Besides cultivated plants, many weed and wild hosts act as virus reservoirs where recombination may occur, resulting in new species. The aim of this study was to further characterise the diversity of begomoviruses infecting two major weed genera, Sida and Leonurus. Total DNA was extracted from samples collected in the states of Rio Grande do Sul, Paraná and Mato Grosso do Sul during the years 2009–2011. Viral genomes were enriched by rolling circle amplification (RCA), linearised into unit length genomes using various restriction enzymes, cloned and sequenced. A total of 78 clones were obtained: 37 clones from Sida spp. plants and 41 clones from Leonurus sibiricus plants. Sequence analysis indicated the presence of six bipartite begomovirus species and two alphasatellites. In Sida spp. plants we found Sida micrantha mosaic virus (SiMMV), Euphorbia yellow mosaic virus (EuYMV), and three isolates that represent new species, for which the following names are proposed: Sida chlorotic mottle virus (SiCMoV), Sida bright yellow mosaic virus (SiBYMV) and Sida golden yellow spot virus (SiGYSV), an Old World‐like begomovirus. L. sibiricus plants had a lower diversity of begomoviruses compared to Sida spp., with only Tomato yellow spot virus (ToYSV) and EuYMV (for the first time detected infecting plants of the genus Leonurus) detected. Two satellite DNA molecules were found: Euphorbia yellow mosaic alphasatellite, for the first time detected infecting plants of the genus Sida, and a new alphasatellite associated with ToYSV in L. sibiricus. These results constitute further evidence of the high species diversity of begomoviruses in non‐cultivated hosts, particularly Sida spp.     

Occurrence of Six Begomoviruses Infecting Tomato Fields in Venezuela and Genetic Characterization of Potato Yellow Mosaic Virus Isolates

Begomoviruses are one of the major pathogens in tomato crops worldwide. In Venezuela, six begomovirus species have been described infecting tomato: Potato yellow mosaic virus (PYMV), Euphorbia mosaic Venezuela virus (EuMVV), Merremia mosaic virus (MeMV), Tomato chlorotic leaf distortion virus (ToCLDV), Tomato yellow margin leaf curl virus (TYMLCV) and Tomato yellow leaf curl virus (TYLCV). In this study, the occurrence of these viruses was analysed by PCR in 338 tomato plants exhibiting virus‐like symptoms. Sixty‐three per cent of the plants were positive at least to one of the begomoviruses tested. PYMV and TYLCV were the most frequent viruses showing 39.6 and 23.7% occurrence, respectively. Phylogenetic analyses revealed two groups of PYMV isolates from several Caribbean Basin countries. The first group clustered isolates from several countries, including Venezuela, and the second group clustered only Colombian isolates. Due to the high prevalence of PYMV and TYLCV in Venezuela, it is suggested that the surveillance and control strategies currently applied in the country should be focused on these two begomoviruses.     

The incidence of viruses in wild Brassica nigra in Dorset (UK)

Using enzyme‐linked immunosorbent assays, the frequency of occurrence of six viruses was determined in Brassica nigra collected from five coastal sites in Dorset, spanning approximately 24 km. During 1998–2000, the viruses detected were: Turnip mosaic virus (genus Potyvirus) (TuMV), Turnip yellow mosaic virus (genus Tymovirus) (TYMV), Turnip crinkle virus (genus Carmovirus) (TCV), Turnip rosette virus (genus Sobemovirus) (TRoV), Beet western yellows virus (genus Polerovirus) (BWYV) and Cauliflower mosaic virus (genus Caulimovirus) (CaMV). Multiple infections were detected in some individuals (48/447). TuMV was detected infrequently over the three‐year period (5/597). A representative isolate of each virus was tested for its effects on glasshouse‐grown individuals from different half‐sib families of B. nigra from four of the sites. Whether inoculated manually or via aphids (Myzus persicae), TuMV caused a rapid (within 10 days) lethal systemic necrosis in the B. nigra seedlings except when they were near flowering at the time of inoculation. Each of the other viruses invaded systemically but were not lethal. Indeed, BWYV systemically invaded 13/19 glasshouse‐grown B. nigra seedlings but did not produce any visible symptoms. Otherwise, the isolates tested differed in their pathogenicity and in the symptoms they produced in infected B. nigra. With TYMV or TCV viral antigen concentration was closely linked to pathogenicity; for TRoV or CaMV, there was little or no difference in virus concentration between plants with and without symptoms. Substantial and reproducible differences were observed in sensitivity/susceptibility among B. nigra genotypes from different sites in Dorset challenged with the same virus isolate.     

Zucchini Yellow Fleck Virus is an Emergent Virus on Melon in Sicily (Italy)

Since 2006, winter melon plants (Cucumis melo L. var inodorus) showing symptoms of pin‐point yellow spots were noticed in Sicily (Italy). Leaf samples were tested by enzyme‐linked immunosorbent assay to the most important viruses‐infecting cucurbits. Zucchini yellow fleck virus (ZYFV, genus Potyvirus) was the only virus detected. Surveys in 2007 and 2008 revealed an increasing number of sites in Sicily with ZYFV‐infected winter melon plants. To confirm the identity of the virus as ZYFV, two isolates from different locations were sequenced and shown to be approximately 85% identical to the published sequences of isolates previously identified in Italy and France. This is the first report of ZYFV occurring on melon in Italy.     

Selective constraints, molecular recombination structure and phylogenetic reconstruction of isometric plant RNA viruses of the families Luteoviridae and Tymoviridae

In an effort to enhance the knowledge on molecular evolution of currently the known members of the families Luteoviridae and Tymoviridae, in-depth molecular investigations in the entire genome of 147 accessions retrieved from the international databases, were carried out. Two algorithms (RECCO and RDP version 3.31β) adapted to the mosaic structure of viruses were utilized. The recombination frequency along the sequences was dissected and demonstrated that the three virus genera of the family Luteoviridae comprise numerous members subjected to recombination. It has pointed out that the major viruses swapped a few but long genomic segments. In addition, in Barley yellow dwarf virus, heredity material might be exchanged between two different serotypes. Even more, putative recombination events occurred between two different genera. Based on Fisher’s Exact Test of Neutrality, positive selection acting on protein expression was detected only in the poleroviruses Cereal yellow dwarf virus, Potato leafroll virus and Wheat yellow dwarf virus. In contrast, several components of the family Tymoviridae were highly recombinant. Genomic portion exchange arose in many positions consisting of short fragments. Furthermore, no positive selection was detected. The evolutionary history showed, in the Luteoviridae, that all screened isolates split into three clusters corresponding to the three virus genera forming this family. Moreover, in the serotype PAV of Barley yellow dwarf virus, two major clades corresponding to PAV-USA and PAV-China, were delineated. Similarly, in the Tymoviridae, all analyzed isolates fell into four groups corresponding to the three virus genera composing this family along with the unclassified Tymoviridae. Inferred phylogenies reshuffled the existing classification and showed that Wheat yellow dwarf virus-RPV was genetically closely related to Cereal yellow dwarf virus and the unclassified Tymoviridae Grapevine syrah virus-1 constituted an integral part of the genus Marafivirus.     

Whitefly‐transmitted RNA viruses that affect intensive vegetable production

Intensive vegetable production is constantly facing the emergence of new viral diseases. Apart from the intrinsic features of viruses as plant pathogens, the highly dynamic turnover of cultivars and cultural practices, and the global trade of seeds and products characteristic of intensive vegetable production may favour the emergence of new viruses, as well as the expansion of the geographical range of vectors responsible for their dissemination. Indeed, the efficient transmission of viruses plays a major role in the impact and outcome of viral epidemics. Whiteflies (Hemiptera: Aleyrodidae) that belong to the genera Bemisia and Trialeurodes are efficient virus vectors. Whiteflies transmit viruses of at least four genera, one of DNA viruses, the genus Begomovirus, and three of RNA viruses, Crinivirus, Ipomovirus and Torradovirus. Begomoviruses have been the subject of recent reviews. In this article we review the genome structure, epidemiology and control of whitefly‐transmitted RNA viruses that belong to the genera Crinivirus, Ipomovirus and Torradovirus, with an extended discussion on the particular viruses within these genera that are currently causing important outbreaks, such are Cucumber vein yellowing virus (CVYV), Cucurbit yellow stunting disorder virus (CYSDV), Tomato chlorosis virus (ToCV) and Tomato torrado virus (ToTV).     

Genome organization and variation in the 3′-partial sequence of garlic latent virus in China

Ten different isolates of a carlavirus were detected by degenerate PCR from 12 garlic samples collected from 6 provinces in China, and the complete genome sequence of the Zhejiang isolate ZJ1 and 3’-terminal sequences of 9 other isolates were determined. The RNA genome of isolate ZJ1 consisted of 8363nts excluding the 3’-poly (A) tail, and the genome organization was similar to other carlaviruses with 6 open reading frames encoding a replicase, TGB1, TGB2, TGB3, CP and NABP respectively. Sequence comparisons showed that all 10 isolates were Garlic latent virus (GarLV). The variations in the TGB2, TGB3 and NABP were more significant than those in the CP. High homology was also detected between those isolates and Shallot latent virus (ShLV). Phylogenetic analysis suggested that GarLV isolates from garlic can be divided into 4 main groups and Chinese isolates belonged to each group. This is the first reported molecular analysis of members of the genus Carlavirus in China.     

Comparative cytopathology of Crinivirus infections in different plant hosts

We used transmission electron microscopy to compare the cytopathology induced in plants by five criniviruses (genus Crinivirus; Lettuce infectious yellows virus (LIYV), Cucurbit yellow stunting disorder virus (CYSDV), Tomato infectious chlorosis virus (TICV), Tomato chlorosis virus (ToCV) and Beet pseudo‐yellows virus (BPYV) (Hartono et al., 2003)). We also compared the patterns of infection for plants and mesophyll protoplasts infected by LIYV and Beet yellows virus (BYV), type members of genera Crinivirus and Closterovirus, respectively. The main cytopathological effects induced in plants by criniviruses were common in young leaves and included alterations of the chloroplasts and the presence of BYV‐type inclusion bodies in companion cells. Virus‐like particles were present in sieve tubes and vascular parenchyma cells as scattered particles, or in companion cells as large masses forming cross‐banded inclusions. Depending on the virus and the plant, it was possible to find virions or virus‐like particles out of the phloem cells, but only in cells of the bundle sheath. Virion‐like particles were never found outside of the vascular tissue. Accumulation of electron‐dense material at the plasmalemma was common for criniviruses, but only LIYV infections produced characteristic conical electron‐dense plasmalemma deposits (PDs). The LIYV‐induced PDs have a crystalline‐like structure and were found at the internal side of plasmalemma.     

Tomato yellow leaf curl viruses: ménage à trois between the virus complex,the plant and the whitefly vector

Tomato yellow leaf curl disease (TYLCD) is one of the most devastating viral diseases affecting tomato crops in tropical, subtropical and temperate regions of the world. Here, we focus on the interactions through recombination between the different begomovirus species causing TYLCD, provide an overview of the interactions with the cellular genes involved in viral replication, and highlight recent progress on the relationships between these viruses and their vector, the whitefly Bemisia tabaci. Taxonomy: The tomato yellow leaf curl virus‐like viruses (TYLCVs) are a complex of begomoviruses (family Geminiviridae, genus Begomovirus) including 10 accepted species: Tomato yellow leaf curl Axarquia virus (TYLCAxV), Tomato yellow leaf curl China virus (TYLCCNV), Tomato yellow leaf curl Guangdong virus (TYLCGuV), Tomato yellow leaf curl Indonesia virus (TYLCIDV), Tomato yellow leaf curl Kanchanaburi virus (TYLVKaV), Tomato yellow leaf curl Malaga virus (TYLCMalV), Tomato yellow leaf curl Mali virus (TYLCMLV), Tomato yellow leaf curl Sardinia virus (TYLCSV), Tomato yellow leaf curl Thailand virus (TYLCTHV), Tomato yellow leaf curl Vietnam virus (TYLCVNV) and Tomato yellow leaf curl virus(TYLCV). We follow the species demarcation criteria of the International Committee on Taxonomy of Viruses (ICTV), the most important of which is an 89% nucleotide identity threshold between full‐length DNA‐A component nucleotide sequences for begomovirus species. Strains of a species are defined by a 93% nucleotide identity threshold. Host range: The primary host of TYLCVs is tomato (Solanum lycopersicum), but they can also naturally infect other crops [common bean (Phaseolus vulgaris), sweet pepper (Capsicum annuum), chilli pepper (C. chinense) and tobacco (Nicotiana tabacum)], a number of ornamentals [petunia (Petunia×hybrida) and lisianthus (Eustoma grandiflora)], as well as common weeds (Solanum nigrum and Datura stramonium). TYLCVs also infect the experimental host Nicotiana benthamiana. Disease symptoms: Infected tomato plants are stunted or dwarfed, with leaflets rolled upwards and inwards; young leaves are slightly chlorotic; in recently infected plants, fruits might not be produced or, if produced, are small and unmarketable. In common bean, some TYLCVs produce the bean leaf crumple disease, with thickening, epinasty, crumpling, blade reduction and upward curling of leaves, as well as abnormal shoot proliferation and internode reduction; the very small leaves result in a bushy appearance.     

Occurrence and Genome Analysis of Cucurbit chlorotic yellows virus in Iran

In 2011 and 2012, several cucurbit‐growing regions of Iran were surveyed and samples with symptoms similar to those induced by Cucurbit chlorotic yellows virus (CCYV) were collected. The pathogen was transmitted to cucumber and melon under greenhouse conditions by whiteflies (Bemisia tabaci). RT‐PCR using designed CCYV‐specific primer pair (CCYV‐F/CCYV‐R) resulted in amplification of the predicted size DNA fragment (870 bp) for the coat protein (CP) gene in samples collected from Boushehr, Eyvanakay and Varamin. Nucleotide sequences of the CP of the three Iranian CCYV isolates were compared with five CCYV isolates obtained from GenBank and analysed. Phylogenetically, all CCYV isolates clustered in two groups; Group I is composed of five non‐Iranian isolates from China, Lebanon, Japan, Sudan and Taiwan, and the three Iranian isolates formed Group 2. Among Iranian isolates, the Eyvanakay isolate clustered in a distinct clade with the Boushehr and Varamin isolates. A phylogenetic tree based on amino acid identity of CP showed that CCYV was closely related to Lettuce chlorosis virus (LCV), Bean yellow disorder virus (BnYDV) and Cucurbit yellow stunting disorder virus (CYSDV). This is the first report of CCYV in Iran.     

Relative incidence,spatial distribution and genetic diversity of cucurbit viruses in eastern Spain

Viral diseases that could cause important economic losses often affect cucurbits, but only limited information on the incidence and spatial distribution of specific viruses is currently available. During the 2005 and 2006 growing seasons, systematic surveys were carried out in open field melon (Cucumis melo), squash and pumpkin (Cucurbita pepo), watermelon (Citrullus lanatus) and cucumber (Cucumis sativus) crops of the Spanish Community of Valencia (eastern Spain), where several counties have a long standing tradition of cucurbit cultivation and production. Surveyed fields were chosen with no previous information as to their sanitation status, and samples were taken from plants that showed virus‐like symptoms. Samples were analysed using molecular hybridisation to detect Beet pseudo‐yellows virus (BPYV), Cucurbit aphid‐borne yellows virus (CABYV), Cucumber mosaic virus (CMV), Cucumber vein yellowing virus (CVYV), Cucurbit yellow stunting disorder virus (CYSDV), Melon necrotic spot virus (MNSV), Papaya ring spot virus (PRSV), Watermelon mosaic virus (WMV) and Zucchini yellow mosaic virus (ZYMV). We collected 1767 samples from 122 independent field plots; out of these, approximately 94% of the samples were infected by at least one of these viruses. Percentages for the more frequently detected viruses were 35.8%, 27.0%, 16.5% and 7.2% for CABYV, WMV, PRSV and ZYMV, respectively, and significant deviations were found on the frequency distributions based on either the area or the host sampled. The number of multiple infections was high (average 36%), particularly for squash (more than 57%), with the most frequent combination being WMV + PRSV (12%) followed by WMV + CABYV (10%). Sequencing of WMV complementary DNA suggested that ‘emerging’ isolates have replaced the ‘classic’ ones, as described in southern regions of France, leading us to believe that cucurbit cultivation could be severely affected by these new, emerging isolates.     

Begomoviruses Associated with Leaf Curl Disease of Tomato in Java,Indonesia*

We report that several begomoviruses are associated with tomato leaf curl disease in Java, Indonesia. Tomato plants with leaf curl symptoms were collected from Bandung (west Java), Purwokerto (central Java), Magelang (central Java) and Malang (east Java) of Indonesia, the major tomato‐growing areas of the country. Viruses were detected using the polymerase chain reaction (PCR), with universal primers for the genus Begomovirus. PCR‐amplified fragments were cloned and sequenced. Based on sequence comparisons and phylogenetic analyses, the viruses were divided into three groups. With respect to amino acid (aa) identities of the N‐terminal halves of the coat proteins compared in this study, group I was most closely related to Ageratum yellow vein virus (AYVV) (97%), Ageratum yellow vein China virus‐[Hn2] (AYVCNV‐[Hn2]) (96%) and Ageratum yellow vein virus‐[Taiwan] (AYVV‐[Tai]) (95%), and ageratum‐infecting begomovirus from Java (99%). Group II had high sequence identity with a tentative species of tomato leaf curl Java virus (ToLCJAV) (96% aa) for the CP. Group III was most closely related to a proposed species of Pepper yellow leaf curl Indonesia virus (PepYLCIDV) (90% aa identity) by its partial CP sequence.     

Isolation and Characterization of Viruses Present in Four Clones of Garlic (Allium sativum) in Venezuela

Isolation and characterization of viruses infecting garlic in Venezuela indicate the presence of onion yellow dwarf virus [OYDV] in most field-grown plants of the clones Criollo venezolano, Morado peruano and Blanco, while 14% of the Criollo venezolano plants showed co-infection of leek yellow stripe virus [LYSV] and OYDV. In leaf tissues OYDV showed an average concentration of 165 μg/g fresh wt., the value being similar among clones. In Criollo venezolano plants co-infected by LYSV and OYDV the concentration of the former was three fold lower than that of OYDV. Serological tests on leaf extracts of garlic plants indicated the absence of the garlic yellow streak virus [GYSV], shallot latent virus [SLV] and garlic latent virus [GLV] in the clones Criollo venezolano, Morado peruano and Blanco. GLV was found to infect plants of the clone Gigante. Physicochemical analysis of purified GLV strongly supports the inclusion of this virus as a definitive member of the carlavirus group.