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.     

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.     

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.     

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。     

Improved Detection of Citrus psorosis virus and Coat Protein‐Derived Transgenes in Citrus Plants: Comparison Between RT‐qPCR and TAS‐ELISA

Citrus is one of the most economically important fruit crops in the world. Citrus psorosis is a serious disease affecting mainly oranges and mandarins in Argentina and Uruguay. The causal agent is Citrus psorosis virus (CPsV), an ophiovirus with a tripartite ssRNA genome of negative polarity. The coat protein (CP), the most abundant viral protein in infected plants, has been used to detect CPsV by TAS‐ELISA, but only biological indexing, requiring 1 year, is the current and validated technique for diagnosis of citrus psorosis. In this study, a SYBR Green RT‐qPCR protocol was developed, with primers designed to the most conserved region of the cp gene. We tested their specificity and sensitivity in comparison with TAS‐ELISA. This RT‐qPCR was applied successfully to field samples from Argentina, to a variety of isolates from different countries maintained in the greenhouse, to young seedlings and old trees from a psorosis natural transmission plot, and to transgenic citrus expressing the cp gene of CPsV or a fragment thereof. This method allowed accurate quantification of viral titer and cp gene expression in transgenic plants, which could not be detected previously. The sensitivity and reliability of quantitative CPsV detection were improved with greater speed using commercial reagents, and the sensitivity was three orders of magnitude higher than that of TAS‐ELISA. All these data encourage its validation.     

One‐step Multiplex RT‐PCR for Simultaneous Detection of Four Viruses in Tobacco

A one‐step multiplex RT‐PCR method has been developed for the simultaneous detection of four viruses frequently occurring in tobacco (Cucumber mosaic virus, Tobacco mosaic virus, Tobacco etch virus and Potato virus Y). Four sets of specific primers were designed to work with the same reaction reagents and cycling conditions, resulting in four distinguishable amplicons representative of the four viruses independently. This one‐step multiplex RT‐PCR is consistently specific using different combinations of virus RNA as templates, and no non‐specific band was observed. It has high sensitivity compared to single RT‐PCR. Moreover, field samples in China can be tested by this method for virus detection. Our results show that one‐step multiplex RT‐PCR is a high‐throughput, specific, sensitive method for tobacco virus detection.     

Detection of Olive‐infecting Viruses in Tunisia

During a virus survey in autumn 2007 and spring 2008 of two Tunisian olive mother blocks, 175 olive samples were collected from 19 different cultivars and tested by RT‐PCR for the presence of Arabis mosaic virus (ArMV), Cherry leaf roll virus (CLRV), Cucumber mosaic virus (CMV), Olive latent ringspot virus (OLRSV), Olive latent virus 1 (OLV‐1), Olive latent virus 2 (OLV‐2), Olive leaf yellowing‐associated virus (OLYaV) and Strawberry latent ringspot virus (SLRSV), using specific sets of primers. The PCR‐negative samples were also subjected to dsRNA and mechanical transmission tests. PCR results indicated that c. 86% of the trees were infected with at least one virus, whereas visible bands were shown by 3 of 24 PCR‐negative samples in dsRNA analysis. OLYaV was the most prevalent virus (49.1%), followed by OLV‐1 (34.3%), CMV (25.7%), OLRSV (16.6%), CLRV (13.1%), SLRSV (7.4%) and OLV‐2 (6.9%), whereas ArMV was not detected. Very high infection rates were found in the two main oil cvs. Chemlali (84.6%) and Chétoui (86.9%).     

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.     

Specific and Rapid Detection of Lily symptomless virus and Arabis mosaic virus in Lily by Dual IC‐RT‐PCR

Lily symptomless virus (LSV) and Arabis mosaic virus (ArMV) cause severe losses of quantity and quality of lily flower and bulb production. Specificity, sensitivity and speed of detection methods for viruses need to be improved greatly to prevent LSV and ArMV from spreading from infected lilies. A dual IC‐RT‐PCR procedure for detection was developed in which the antibodies of LSV and ArMV were mixed and the mixture used to coat the PCR tubes. The particles of the two viruses were captured by the respective antibodies. Interference by other RNA viruses in infected lily was eliminated in the RT‐PCR. Also, an RNA extraction step was omitted. The dual IC‐RT‐PCR products of LSV and ArMV were 521 bp and 691 bp, respectively. The specificity of the method was validated; only LSV and ArMV of four viruses were detected by dual IC‐RT‐PCR. The sensitivity of the detection method is 1 mg leaf tissue and higher than DAS‐ELISA due to enrichment by dual immunocapture.