Improved PCR detection of potyviruses in Allium species

Protocols for producing virus-free Allium plants require an indexing system that is more sensitive than DAS-ELISA and can detect low virus concentrations in infected plants. In the present work, degenerate primers were designed and a one-step IC-RT-PCR protocol was developed to differentiate between Leek yellow stripe virus (LYSV) and Onion yellow dwarf virus (OYDV) in single and mixed infections in several Allium spp. A 566-bp band was observed for LYSV, a 489-bp band for OYDV in single infections, and two bands of the same sizes in mixed infections in different species of Alliaceae. A 508-bp band of Shallot yellow stripe virus and a 594-bp band of Turnip mosaic virus were also amplified with the same primers. RT-nested-PCR was also conducted directly in microtitre plate wells after negative or questionable reactions were produced in an ELISA experiment. The detection limit of the DAS-ELISA for LYSV was 16.5-27.3 ng ml-1. The RT-nested-PCR done after DAS-ELISA was 102 times more sensitive than the DAS-ELISA alone. In parallel, an IC-RT-nested-PCR in microcentrifuge tubes was 104 times more sensitive than the DAS-ELISA. The DAS-ELISA-RT-nested-PCR enables the initial screening of samples by DAS-ELISA to eliminate a high percentage of virus-positive plants, considerably reducing the number of plants to analyze further by RT-PCR. © Springer 2005

A biomarker for the identification of four Phaeoacremonium species using the β-tubulin gene as the target sequence

A real-time polymerase chain reaction (PCR) was developed for the rapid detection and identification of Phaeoacremonium species, the fungi associated with severe diseases in grapevines. A degenerate primer pair (F2bt-R1bt) with homology to the β-tubulin gene was designed to be used in the amplification of 11 species of Phaeoacremonium. Four species-specific probes labelled with three different fluorescent dyes were designed to be used with the degenerate primers in a real-time PCR for the identification of Phaeoacremonium aleophilum, P. parasiticum, P. viticola and P. mortoniae. Combinations of two probes in a duplex real-time PCR allowed to detect and identify a mixture of Phaeoacremonium species and cross-amplifications were not detected. This method was applied to detect Phaeoacremonium species in eight wood fragments from grapevine plants naturally infected, and results were compared with those obtained with nested PCR and culturing on growth media. Real-time PCR detected Phaeoacremonium in 100% of the analysed fragments, whereas nested PCR did only in the 62% of them and requiring subsequent restriction fragment-length polymorphism analysis to identify the species. This method is a sensitive tool to detect and identify Phaeoacremonium species in infected grapevine wood. Real-time PCR assay defined here can be used in a plant nursery program to identify pathogen-free plants in order to manage Petri disease of grapevines. © 2008 Springer-Verlag

Plant viral elongated nanoparticles modified for log-increases of foreign peptide immunogenicity and specific antibody detection

Elongated and flexuous recombinant nanoparticles were derived from Turnip mosaic virus to be used as bioscaffolds for increased peptide immunogenicity and peptide-specific antibody sensing. For this purpose, a 20-amino acid peptide derived from human vascular endothelial growth factor receptor 3 (VEGFR-3) was fused to the N-terminal region of Turnip mosaic virus coat protein (CP) by genetic insertion. The insertion was between codons corresponding to the first and second amino acids of the CP in two versions of a previously reported virus-derived vector. Systemic infections of two genetic constructs were achieved in two different plant hosts. The construct proved stable upon successive passages and generated virus nanoparticles identifiable under the electron microscope. The chimeric structures held the VEGFR-3 peptide. Purified VER3 nanoparticles were used to immunize mice, whose sera showed log increases of antibodies against the VEGFR-3 peptide when compared with mice immunized with peptide alone, thus providing the first quantitative data on the potential of elongated flexuous viruses for peptide immunogenicity increases. Purified VER3 nanoparticles also showed log increases in their ability to detect VER3 antibodies in sera, when used as reagents in ELISA assays, an application also used here for the first time. © 2013 Elsevier B.V

A developmentally linked, dramatic, and transient loss of virus from roots of Arabidopsis thaliana plants infected by either of two RNA viruses

Possible effects of host developmental stage on the amount of virus present in systemically infected plant tissues hitherto have received little attention. In this study, the pattern of virus accumulation over the plant lifespan has been examined in systemically invaded tissues of Arabidopsis thaliana infected by either of two distinct (+)RNA viruses Turnip mosaic virus, a member of Potyvirus, and Oilseed rape mosaic virus, a member of Tobamovirus. Quantitative analyses of virus coat protein and virus genomic RNA in roots versus aerial plant parts revealed generally sinusoidal temporal patterns of virus accumulation. In noninoculated leaves, a time period was found during which no virus accumulation was detected. This period was coincident with the approximately 7 days of inflorescence bud formation and differentiation. In roots, virion content reached high levels a few days after inoculation, dropping dramatically during the period of bud formation and quickly recovering after it. These results, together with electron microscopy observations, are consistent with loss of virions due to disassembly. Fluorescence observations of green fluorescent protein-tagged virus-infected root tissue also were consistent with a net loss of virus-specified proteins. Inoculations performed after the emergence of the inflorescence and on A. thaliana flowering-time mutants support the temporal link between observed changes in virus content and inflorescence bud formation. Different host-involving biochemical processes can be invoked to provide mechanistic clues, but no one of them alone seems sufficient to explain the complex patterns of tight temporal regulation of virus accumulation observed in these experiments. © 2007 The American Phytopathological Society

Ultra-sensitive detection of two garlic potyviruses using a real-time fluorescent (Taqman®) RT-PCR assay

A method for the detection of Onion yellow dwarf virus (OYDV) and Leek yellow stripe virus (LYSV), the two most prevalent garlic potyviruses, has been developed that combines IC-RT-PCR/RT-PCR with the use of TaqMan® probes. Comparisons with ELISA results obtained with identical OYDV and LYSV infected samples showed sensitivity in detecting these viruses increased up to 106-fold. OYDV and LYSV were detected using different fluorochromes in the probe, thus allowing unequivocal diagnosis for each of them. The polyvalence of the designed virus-specific primers and probes was shown through their application to the detection of three isolates from very different geographical areas and from different hosts. A second version of the method avoids the need for an immunocapture step through the performance of a TaqMan® RT-PCR assay directly on extracts of garlic cloves. This modification on the proposed basic method allows the analysis of bulb samples in 3-4 h but did not give reproducible results with leaves. Both versions of the new diagnostic method bear great potential for their implementation in virus-free certification schemes in garlic, a vegetatively propagated crop for which such a certification is critical for a high-quality product. © 2004 Elsevier B.V. All rights reserved

Genomic heterogeneity and host recovery of isolates of Malva vein clearing virus

Malva vein clearing virus (MVCV), a tentative species of the genus Potyvirus, was identified as the causal agent of viral symptoms in Malva sp. weed plants. Amplified viral genomic fragments corresponding to approximately 20% of the 3′ terminal region of its genome were obtained using non-species specific, genus-specific reagents. The sequences of the PCR fragments were determined. BLAST and phylogenetic analyses of the deduced amino acid sequence indicated that MVCV is a distinct species of the genus Potyvirus and close to Pea seed-borne mosaic virus (PSbMV) with which it forms a new phylogenetic cluster within the genus. The results show that MVCV is a definitive member of the Potyvirus genus. Specific MVCV PCR primers were designed and validated as diagnostic tools, and used to assess the variability of the species. Much variation was found and this was not correlated with either the geographical origin of the isolates, or the severity of the symptoms. Recovery from viral symptoms was observed in natural and experimental hosts. Tests in experimental hosts showed that it was a true viral recovery, in that the virus was absent and the recovered tissues could not be infected. This is the first reported example of true viral recovery of a potyvirus in a natural system. © 2008 Elsevier B.V. All rights reserved

Disturbance of Arabidopsis thaliana development by a potyviral infection maps to the P3/p6k1 viral genomic region

Infections of plants by viruses induce plant disease and associated symptoms result in economic losses in crops. The study of viral infections has led to the discovery of RNA silencing as a plant defence mechanism against plant pathogens and of viral suppressors of gene silencing as the viral mechanism to counter such plant defence. In addition, it has led to the unravelling of the role of small RNAs (sRNAs) in plant development. Developmental symptoms associated with plant disease have been attributed in some systems to the effects of the viral suppressors of gene silencing on the normal performance of the plant sRNA machinery. In the model system Arabidopsis thaliana - Turnip mosaic virus, a potyvirus two different strains of which induce very different disturbances of the plant development, we have identified the viral determinant of developmental symptoms in the P3/p6k1 region, different from the described viral suppressor of gene silencing (HC-Pro). This result emphasises the role of the different viral proteins in disease induction, opens the way to deepen our knowledge of the potyviral proteins in the viral cycle and also to better understand plant growth regulation. Results will be presented and discussed

Allium genetic resources

An overview of the developments in Allium genetic resources during the past 25 years is presented in this chapter. A first important development has been the introduction and further development of web-based genebanking information systems (e.g. GENESYS, PLANTSEARCH), which facilitated the exchange of data to a large extent between Allium collection holders worldwide. These information systems made it possible to obtain an overview of the Allium genetic resources managed worldwide and identify the gaps in collections which still need to be filled, especially in the face of the ongoing genetic erosion. A second important area of progress has been the development of new methods for the maintenance of Allium germplasm, especially cryopreservation. This method has made it possible to maintain Allium accessions in a cheap and effective way. The method is especially important for the conservation of vegetatively maintained germplasm. Other developments in Allium genebanking are the improvement of the health status of the germplasm kept in the collections and the continuing characterization and evaluation of germplasm, which stimulates the utilization of the Allium genetic resources held in genebanks. Significant changes could also be observed with respect to acquisition and exchange of plant genetic resources due to many and complex new regulations on the legal and organizational levels due to the adoption of the CBD and IT-PGRFA by many countries. It makes the handling of the plant accessions safer and more consistent but also more circumstantial. Finally, we need to underline that in an increasingly changing world with all the threats of genetic erosion and extinction due to disappearance of traditional cultivation methods, devastation of our environment and climatic change, the conservation of genetic resources is of prime importance for agriculture. Especially for breeders, a highly diverse genepool of a crop plant is an invaluable treasure. The importance to keep this treasure will no doubt become even more important in the future