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.     

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.     

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.     

韭葱黄条病毒、洋葱黄矮病毒和胡葱黄条病毒六安分离物CP基因克隆及同源性分析

设计特异性引物PCR扩增了六安大蒜病样中的韭葱黄条病毒(Leek yellow stripe virus,LYSV)、洋葱黄矮病毒(Onion yellow dwarf virus,OYDV)和胡葱黄条病毒(Shallot yellow stripe virus,SYSV)的全长CP基因,插入到pGEM-T载体并测序。分别比较3种病毒CP基因种内变异性和种间亲缘关系。结果表明LYSV六安分离物CP基因由864个碱基组成,与Genbank上已报道的68个LYSV不同分离物CP基因的核苷酸序列同源性为76.12%～84.31%;OYDV的CP基因由771个碱基组成,与Genbank上已报道的86个OYDV不同分离物同源性为81.06%～90.40%;SYSV的CP基因由774个碱基组成,与Genbank上已报道的11个SYSV不同分离物CP基因同源性为88.63%～94.32%;从分析结果来看,LYSV的CP基因不同分离物之间变异性较大,OYDV CP变异性不大,SYSV变异性很小;3种病毒都有1个以上的宿主,病毒种内不同宿主分离物之间CP序列差异很小。进化分析显示OYDV和SYSV的CP基因亲缘性较近并成簇,LYSV的CP基因与OYDV和LYSV的CP基因亲缘性较远。     

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.     

Complete genome sequence of garlic latent virus,a member of the carlavirus family

The complete genome sequence of the garlic latent virus (GLV) has been determined. The whole GLV genome consists of 8,353 nucleotides, excluding the 3'-end poly(A)+ tail, and contains six open-reading frames (ORFs). Putative proteins that were encoded by the reading frames contain the motifs that were conserved in carlavirus-specific RNA replicases, NTP-dependent DNA helicases, two viral membrane-bound proteins, a viral coat protein, and a zinc-finger. Overall, the GLV genome shows structural features that are common in carlaviruses. An in vitro translation analysis revealed that the zinc-finger protein is not produced as a transframe protein with the coat protein by ribosomal frameshifting. A Northern blot analysis showed that GLV-specific probes hybridized to garlic leaf RNA fragments of about 2.6 and 1.5 kb long, in addition to the 8.5 kb whole genome. The two subgenomic RNAs might be encapsidated into smaller viral particles. In garlic plants, 700 nm long flexuous rod-shaped virus particles were observed in the immunoelectron microscopy using polyclonal antibodies against the GLV coat proteins.     

Efficient elimination of virus complex from garlic (<Emphasis Type="Italic">Allium sativum</Emphasis> L.) by cryotherapy of shoot tips

Vegetative propagation of plants, such as garlic (Allium sativum L.), is known to facilitate the transmission of several virus species throughout the plant cycles. This process favors the onset of complex diseases by accumulation of different species in the same plant, resulting in decreased productivity and production quality. Studies have reported the use of cryotherapy of shoot tips, or meristematic clusters, as an efficient tool for obtaining virus-free plants. This study aimed to evaluate the ability of cryotherapy to eradicate virus complex in garlic plants. Bulbils naturally infected with Onion Yellow Dwarf Virus (OYDV), Leek Yellow Strip Virus (LYSV) and Garlic Common Latent Virus (GCLV) were employed as explants for different virus-cleaning treatments tested. Dot-ELISA and RT-PCR analysis were used to demonstrate the presence/absence of virus complex, and histological analysis was also performed to confirm these results. Five days after cryotherapy, structural analysis revealed that cooling had caused cell damage, as indicated by the increased vacuolization of cells after cryotherapy, as well as slight plasmolysis after thermotherapy. Immunolocalization analysis indicated the subcellular distribution of OYDV in garlic shoot tips in association with the development of plasmodesmata, while no OYDV was detected in the first cell layers of the meristematic dome. Cryotherapy successfully removed virus complex, resulting in virus-free plants with enhanced efficiency, compared to conventional meristem culture-based techniques. Moreover, the synergistic effects of cryotherapy and thermotherapy resulted in a 40 % survival rate of shoot tips and the regeneration of 90, 100 and 80 % OYDV-, LYSV- and GCLV-free plants, respectively.     

Virus Free Garlic (Allium Sativum L.) Plants Obtained by Thermotherapy and Meristem Tip Culture

Virus infection in garlic considerably reduces yield and quality in Argentina. The production of virus free “seed” was attempted by means of thermotherapy and meristem tip culture. A hot water treatment was employed to determine the lethal temperature/time combination for clonal type (c.t.) Blanco cloves. It was established that 50°C × 20 min, 50°C × 15 min and 55°C × 5 min were the limit thermal/time combinations which garlic could withstand. Those treatments were employed followed by meristem tip culture, however, none of the successfully developed plants after culture (only 13 %) were virus-free. Hot air treatments in a growth chamber at 36°C lasting for 30, 40 and 60 days, and at 25°–32° for 30 days in a greenhouse were tested on c.t. Blanco. Cloves kept at room temperature throughout the experiment were employed as controls. In the 25°–32°C treatment, 73% of meristems produced plants and, of these, 33 % were virus free. After 30 and 40 days at 36 °C, 62 % and 67 % of the meristems developed into plantlets, of which respectively 51 % and 50 % were virus-free. Very few meristems (10 %) developed into plants when cloves had been kept at 36°C for 60 days but the resulting plantlets were all virus free. Controls produced 78 % of plants, of which 14 % were virus free. Results of hot air treatments of 36 °C for 40 days performed on c.t. Colorado, Rosado, Paraguayo, Espaol and Hilario Ascasubi were similar to those obtained with c.t. Blanco. In Espaol and Hilario Ascasubi, no virus-free plants were detected among control specimens (no thermotherapy treatment). The only virus (from up to 3 that infected the plants) that persisted in some plants after themotherapy and meristem tip culture was garlic yellow streak.     

Determination of Onion yellow dwarf virus concentration levels on onion bulb and leaf by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA)

With the aim of understanding virus movement and fluctuations in the virus concentration in bulb and leaves of onion (Allium cepa L.) plants after infection, Onion yellow dwarf virus (OYDV) was analysed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). OYDV concentrations were higher in onion leaves of plants grown from tested bulbs compared with bulbs, although the virus was successfully detected in bulb of onion.     

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.     

两株大蒜病毒外壳蛋白基因的克隆及其在大肠杆菌中的表达

利用建立cDNA库的方法 ,通过RT PCR分别扩增和克隆了感染我国天津地区大蒜的大蒜花叶病毒 (GMVc)和大蒜潜隐病毒 (GLVc)的外壳蛋白 (CP)基因 ,并对其分别在原核细胞中进行了诱导表达和表达产物的分析。克隆基因的序列测定与分析表明 ,GMVc的CP基因全长 867个核苷酸 ,编码 2 89个氨基酸 ,与报道的一株GMV的核苷酸和氨基酸的同源性分别为 88.5%和 97.2 % ,属于马铃薯Y病毒属 (Potyvirus)的成员 ;GLVc的CP基因全长 885个核苷酸 ,编码 2.94个氨基酸 ,与报道的一株GLV的核苷酸和氨基酸同源率分别为 73.6%和 90.9% ,属于香石竹潜隐病毒属 (Carlavirus)的成员 ;克隆基因的表达产物经SDS PAGE分析表明 ,GM Vc和GLVc的外壳蛋白分别约 32kD和 34kD ,与预测的结果一致。本实验结果对感染我国大蒜的病毒进行分子水平的确切诊断、分类和调查奠定了基础 ,将对大蒜病毒病的防治与脱毒大蒜的生产具有重要的意义。     

Garlic yellow streak virus, a potyvirus infecting garlic in New Zealand

In New Zealand, all garlic (Allium sativum) plants tested were infected by a virus with flexuous filamentous particles 700–800 nm long. This virus, called garlic yellow streak virus (GYSV), infected only two of 12 species tested and was transmitted to garlic by the aphid Myzus persicae in a non-persistent manner. In garlic sap, GYSV was infective at a dilution of 10^-4 but not 10^-3, after heating for 10 min at 60°C but not 65°C, and after 2 days but not 3 days at 25°C. The yield of virus, purified from naturally infected garlic, was 3–4 mg/kg fresh leaf. Preparations had A₂₆₀/A₂₈₀= 1.28 and A_man/A_min= 1.08. The virus particles had a sedimentation coefficient of 149S and a buoyant density in CsCl of 1.334 g/cm³. Mol. wt estimates for the virus nucleic acid were 2.95 × 10⁶ by electrophoresis in polyacrylamide gels and 3.46 × 10⁶ from the sedimentation coefficient (41.4S) in linear-log sucrose density gradients. Two polypeptides were detected in virus preparations; one (mol. wt 30 500) was possibly a breakdown product of the other (mol. wt 33 000). GYSV was serologically distantly related to onion yellow dwarf and leek yellow stripe viruses but was considered to be a separate virus because it differed from them in host range.     

Tête à tête of tomato yellow leaf curl virus and tomato yellow leaf curl sardinia virus in single nuclei

Since 1997 two distinct geminivirus species, Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV), have caused a similar yellow leaf curl disease in tomato, coexisted in the fields of southern Spain, and very frequently doubly infected single plants. Tomatoes as well as experimental test plants (e.g., Nicotiana benthamiana) showed enhanced symptoms upon mixed infections under greenhouse conditions. Viral DNA accumulated to a similar extent in singly and doubly infected plants. In situ tissue hybridization showed TYLCSV and TYLCV DNAs to be confined to the phloem in both hosts, irrespective of whether they were inoculated individually or in combination. The number of infected nuclei in singly or doubly infected plants was determined by in situ hybridization of purified nuclei. The percentage of nuclei containing viral DNA (i.e., 1.4% in tomato or 6% in N. benthamiana) was the same in plants infected with either TYLCSV, TYLCV, or both. In situ hybridization of doubly infected plants, with probes that discriminate between both DNAs, revealed that at least one-fifth of infected nuclei harbored DNAs from both virus species. Such a high number of coinfected nuclei may explain why recombination between different geminivirus DNAs occurs frequently. The impact of these findings for epidemiology and for resistance breeding concerning tomato yellow leaf curl diseases is discussed.     

The synthesis of polyamines from methionine in intact and disrupted leaf protoplasts of virus-infected chinese cabbage

In exploring the role of the chloroplast in the multiplication of turnip yellow mosaic virus, the biosyntheses of the major viral polyamine, spermidine, as well as that of the tetramine, spermine were studied. The synthesis of these polyamines from [2-¹⁴C]methionine in protoplasts of Chinese cabbage leaf cells derived from healthy plants or those infected by turnip yellow mosaic virus were examined. Populations of protoplasts of infected leaves are homogeneous with respect to containing chloroplast aggregates in contrast to those of healthy leaves. Protoplast preparations have been shown to incorporate methionine into protein, spermidine, and spermine more rapidly than do fresh leaf discs, which also show a very slow utilization of labeled arginine and ornithine into polyamine.     

Emission of herbivore-induced volatiles in absence of a herbivore--response of Zea mays to green leaf volatiles and terpenoids

Green leaf volatiles (GLV), a series of saturated and monounsaturated six-carbon aldehydes, alcohols, and esters are emitted by plants upon mechanical damage. Evidence is increasing that intact plants respond to GLV by activating their own defense mechanisms, thus suggesting that they function in plant-plant communication. The present paper demonstrates that exposure of maize plants to naturally occurring GLV, including (Z)-3-, (E)-2- and saturated derivatives, induce the emission of volatile blends typically associated with herbivory. Position or configuration of a double bond, but not the functional group of the GLV influenced the strength of the emissions. (Z)-3-Configured compounds elicited stronger responses than (E)-2- and saturated derivatives. The response to (Z)-3-hexen-1-ol increased linearly with the dose between 200 and 1000 nmol per plant. Not only the naturally occurring (E)-2-hexenal, but also (E)-2-pentenal and (E)-2-heptenal induced maize plants, although to a lesser extent. Externally applied terpenoids [(3E)-4,8-dimethyl-1,3,7-nonatriene, beta-caryophyllene, and (E)-beta-farnesene] did not significantly increase the total amount of inducible volatiles in maize. Of three tested maize cultivars Delprim and Pactol responded much stronger than Attribut. Recovery experiments in the presence and absence of maize plants demonstrated that large proportions of externally applied GLV were assimilated by the plants, whereas (3E)-4,8-dimethyl-1,3,7-nonatriene was recovered in much higher amounts. The results furthermore suggested that plants converted a part of the assimilated leaf aldehydes and alcohols to the respective acetates. We propose that GLV not only can alert neighboring plants, but may facilitate intra-plant information transfer and can help mediate the systemic defense response in a plant.