Complete genome sequence and construction of an infectious full-length cDNA clone of a cucumber vein yellowing virus (CVYV) isolate from Portugal

Cucumber vein yellowing virus (CVYV) is a member of the genus Ipomovirus in the family Potyviridae. In the National Center for Biotechnology Information (NCBI) database, three complete genome sequences of CVYV isolates from Spain (NC_006941), Israel (KT276369), and Jordan (JF460793) are available. In this study, we report the complete sequence of an isolate of CVYV from Portugal (DSMZ PV-0776) along with the construction of an infectious full-length cDNA clone via Gibson assembly. The sequence of CVYV Portugal shows the closest relationship to a CVYV isolate from Spain (genome, 99.7% identity; polyprotein, 99.7% identity). The CVYV full-length cDNA clone was introduced by electroporation into Rhizobium radiobacter and infiltrated into the cotyledons of Cucumis sativus plantlets, resulting in symptoms resembling those of the wild-type virus. Transmission of the infectious CVYV full-length clone by the whitefly Bemisia tabaci was confirmed. This first report confirming the infectivity of a CVYV cDNA clone provides the opportunity to study gene functions in a consistent genomic background

Molecular identification of Nectriaceae in infections of apple replant disease affected roots collected by Harris Uni-Core punching or laser microdissection

Apple replant disease (ARD) negatively affects growth and yield of apple plants worldwide. Fungi belonging to the Nectriaceae have often been isolated from roots grown in replant soils and thus are proposed among others as one biotic cause of the disease complex. Microscopic analyses of ARD-affected roots revealed characteristic symptoms associated with fungal infection sites. Here, two extraction methods of such tissue sites were applied to directly identify an unknown fungus that forms typical cauliflower-like structures in diseased root cortex cells. Punching small tissue samples of about 0.5 mm3 volume with the Harris Uni-Core is a quick and easy method to harvest symptomatic material. Secondly, a laser microdissection (LMD) protocol for apple roots was established. This technique allows the extraction of defined cell or tissue fractions from thin cryo-sections. Tissue harvesting was followed by the identification of fungi via PCR amplification of two gene fragments and Sanger sequencing. For Harris samples, Chelex was used for DNA stabilization, while LMD samples were directly submitted to PCR. In Harris samples, mainly the Nectriaceae species Dactylonectria torresensis, Ilyonectria robusta and Rugonectria rugulosa were identified. In addition to these, in LMD samples Cylindrocladiella sp. and Ilyonectria europaea were detected. Thus, the intracellular CF structures contained different species of Nectriaceae in the ARD-affected cortex cells. These results contribute considerably to the etiology of the ARD. Both protocols offer the possibility to identify fungi from selected symptomatic small root sections by molecular tools avoiding isolation and subsequent axenic pure cultures of single fungal isolates

Author Correction: Observations on early fungal infections with relevance for replant disease in fine roots of the rose rootstock Rosa corymbifera 'Laxa'

The Supplementary Table 2 file published with this Article was incomplete. The Gene Bank Accessions numbers for genes Histone 3 (HIS), partial ß-tubulin (TUB) and translation elongation factor 1-a (TEF) were omitted. The original Supplementary Table 2 file is provided below. This error has now been corrected in the Supplementary Information file that accompanies the original Article

Observations on early fungal infections with relevance for replant disease in fine roots of the rose rootstock Rosa corymbifera 'Laxa'.

Replant disease is a worldwide phenomenon affecting various woody plant genera and species, especially within the Rosaceae. Compared to decades of intensive studies regarding replant disease of apple (ARD), the replant disease of roses (RRD) has hardly been investigated. The etiology of RRD is also still unclear and a remedy desperately needed. In greenhouse pot trials with seedlings of the RRD-sensitive rootstock Rosa corymbifera ‘Laxa’ cultured in replant disease affected soils from two different locations, early RRD symptom development was studied in fine roots. In microscopic analyses we found similarities to ARD symptoms with regards to structural damages, impairment in the root hair status, and necroses and blackening in the cortex tissue. Examinations of both whole mounts and thin sections of fine root segments revealed frequent conspicuous fungal infections in association with the cellular disorders. Particularly striking were fungal intracellular structures with pathogenic characteristics that are described for the first time. Isolated fungi from these tissue areas were identified by means of ITS primers, and many of them were members of the Nectriaceae. In a next step, 35 of these isolates were subjected to a multi-locus sequence analysis and the results revealed that several genera and species were involved in the development of RRD within a single rose plant. Inoculations with selected single isolates (Rugonectria rugulosa and Ilyonectria robusta) in a Perlite assay confirmed their pathogenic relationship to early necrotic host plant reactions, and symptoms were similar to those exhibited in ARD

Advance in grain legumes genetic transformation: The case of GM pea and cowpea

Grain legumes are socio-economically important crops playing a substantial role in providing dietary protein for millions of households in the world. As multipurpose crops, they are used for different purposes such as food and feed. They also fix atmospheric nitrogen contributing to the sustainability of farming system by enriching soil fertility and maintaining the productivity of agricultural land. However, different production factors, such as insect pests and diseases, have limited the productivity of grain legumes both in field and during the storage and are impacting their contribution to nutrition security and poverty reduction. Furthermore, in the current trend of climate change, there is an increasing pressure on plant breeders to develop climate-smart varieties of crops with multiple traits against the different production factors. In order to enhance the economic and social contribution of grain legumes, genetic transformation approaches have been used to develop transgenic lines with new traits such as resistance to insects and diseases as well as tolerance to drought. In this paper, the experience and result of pea and cowpea Agrobacterium-mediated transformation will be presented. Special emphasis will be given to the success and challenges of transgenic insect resistance and its importance in these two important grain legumes. Based on insect bioassay tests, the level of insect resistance in some of the transgenic lines will be presented against that of none transgenic lines. Finally, recommendation will also be discussed for future genetic transformation to develop climate-smart variety of transgenic grain legumes

Endoscopic submucosal dissection with a novel high viscosity injection solution (LiftUp) in an ex vivo model: a prospective randomized study

Introduction  Endoscopic submucosal dissection (ESD) is increasingly being used in the western world. Submucosal injectates are an essential tool for the ESD procedure. In this study, we evaluated a novel copolymer injectate (LiftUp, Ovesco, Tübingen Germany) in an established ESD model (EASIE-R) in comparison to existing submucosal injectables. Materials and methods  We conducted a prospective, randomized ex vivo study performing ESD with three injectates: LiftUp, hydroxyethyl starch (HAES 6 %) and normal saline solution (NaCl 0.9 %). A total of 60 artificial lesions, each 3 × 3 cm in size, were resected in an ex vivo porcine model, utilizing one of the three studied injectates (n = 20 ESDs per injectate). Study parameters were: en bloc resection rate, perforation rate, lifting property, time of injection, injectate volume, general ESD procedure time, and overall procedure time. Results  All 60 lesions were successfully resected using the standard ESD technique. LiftUp had no procedure related perforations, one perforation occurred in the HAES group, and two perforations in the NaCl group ( P  > 0.05). Furthermore, adequate lifting was achieved in 16/20 (80 %) using LiftUp, 6/20 (30 %) in the HAES group and 6/20 (30 %) in the NaCl group ( P  < 0.0002). En bloc resection was achieved in 19 (95 %) with LiftUp, in 20 (100 %) with HAES, and in 16 (80 %) with NaCl. General ESD procedure time and overall procedure time were not different among the three groups. Conclusion  LiftUp appears to be a safe alternative to established fluids for ESD. It had a significantly improved lifting effect and required significantly less injected volume compared to well-established lifting solutions

Peanut yellow spot virus: A distinct tospovirus species based on serology and nucleic acid hybridisation

Nucleocapsids of peanut yellow spot virus (PYSV), purified from peanut (= groundnut) plant tissue, contained a protein with a molecular mass of 29 kDa. In ELISA and immuno-blot analysis the virus did not react with tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV) and peanut bud necrosis virus (PBNV) antisera. PYSV contained three RNA species, a large (L) RNA (c.8900 nucleotides), a medium (M) RNA (c.4800 nucleotides) and a small (S) RNA (c.3000 nucleotides), similar to other tospoviruses. In addition, a fourth RNA species of approximately 1800 nucleotides was also present in purified preparations. Hybridisation analysis under high stringency conditions revealed no hybridisation between PYSV RNAs and cDNA probes representing the nucleocapsid (N) gene, the glycoprotein (GP) gene and the 3' half of the RNA polymerase gene of PBNV. PYSV genomic RNAs also failed to hybridise with cDNA probes from the GP genes of TSWV and INSV. In reciprocal tests, the cDNA clones of PYSV S and M RNAs did not hybridise with any of the PBNV RNAs. Based on the absence of serological relationships between PYSV and PBNV, TSWV and INSV and lack of nucleotide homology based on hybridisation studies between the PYSV RNAs and cDNA clones from PBNV, TSWV and INSV, PYSV should be considered as a distinct species of the genus Tospovirus under a new serogroup, putatively designated ‘V