First report on detection of three Bunya-Like Viruses in apples in Brazil.

Apple chlorotic leaf spot virus (ACLSV), apple stem grooving virus (ASGV), and apple stem pitting virus (ASPV) cause significant losses to Brazilian (BR) apple production. Looking beyond these latent viruses, high-throughput sequencing (HTS) of three samples (Vacaria, Brazil) was performed on an Illumina HiSeq X Ten platform (USA), cv. Braeburn (BB), and a BGISEQ-500 platform (China), cvs. Royal Gala (RG) and Mishima (MI)

Next-Generation Sequencing and Genome Editing in Plant Virology

Next-generation sequencing (NGS) has been applied to plant virology since 2009. NGS provides highly efficient, rapid, low cost DNA, or RNA high-throughput sequencing of the genomes of plant viruses and viroids and of the specific small RNAs generated during the infection process. These small RNAs, which cover frequently the whole genome of the infectious agent, are 21–24 nt long and are known as vsRNAs for viruses and vd-sRNAs for viroids. NGS has been used in a number of studies in plant virology including, but not limited to, discovery of novel viruses and viroids as well as detection and identification of those pathogens already known, analysis of genome diversity and evolution, and study of pathogen epidemiology. The genome engineering editing method, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been successfully used recently to engineer resistance to DNA geminiviruses (family, Geminiviridae) by targeting different viral genome sequences in infected Nicotiana benthamiana or Arabidopsis plants. The DNA viruses targeted include tomato yellow leaf curl virus and merremia mosaic virus (begomovirus); beet curly top virus and beet severe curly top virus (curtovirus); and bean yellow dwarf virus (mastrevirus). The technique has also been used against the RNA viruses zucchini yellow mosaic virus, papaya ringspot virus and turnip mosaic virus (potyvirus) and cucumber vein yellowing virus (ipomovirus, family, Potyviridae) by targeting the translation initiation genes eIF4E in cucumber or Arabidopsis plants. From these recent advances of major importance, it is expected that NGS and CRISPR-Cas technologies will play a significant role in the very near future in advancing the field of plant virology and connecting it with other related fields of biology.Work in RF laboratory has been supported by the Spanish Ministerio de Economía y Competitividad (grant BFU2014-56812-P), and in MB laboratory by Consiglio per la Ricerca in Agricoltura e l’analisi dell’Economia Agraria (CREA).Peer reviewedPeer Reviewe

Complete genomic sequence of Raphanus sativus cryptic virus 4 (RsCV4), a novel alphapartitivirus from radish

The present work reports the discovery and complete genome sequencing of a virus from symptomless radish seedlings, classifiable as a novel member of the genus Alphapartitivirus, family Partitiviridae. Total RNA extracted from germinating seedlings was sequenced using Illumina technology. Bioinformatic analysis of the RNA-seq data revealed two contigs representing the near full-length genomic sequences of two genomic RNAs representing a new virus. Analysis of the genome sequence (excluding the polyA tail, RNA1: 1976 nt and RNA2: 1751 nt, respectively) showed a genomic organization typical of viruses classed within the Partitiviridae, with each genomic RNA encoding a single open reading frame (ORF). Phylogenetic analysis of the RNA dependent RNA polymerase (RNA1 ORF) and of the capsid protein (RNA2 ORF) clearly showed the new virus can be classified within the genus Alphapartitivirus, but sequence divergence establishes it as a new species, for which the name “Raphanus sativus cryptic virus 4” is proposed

Strategies to facilitate the development of uncloned or cloned infectious full-length viral cDNAs: Apple chlorotic leaf spot virus as a case study

Background Approaches to simplify and streamline the construction of full-length infectious cDNA clones (FL-cDNAs) are needed. Among desirable improvements are the ability to use total nucleic acids (TNA) extracts from infected hosts (to bypass viral purification limitations) for the direct one-step amplification of large FL-cDNAs, the possibility to inoculate plants with uncloned FL-cDNAs and the simplified cloning of these large molecules. Results Using the 7.55 kb genome of Apple chlorotic leaf spot trichovirus (ACLSV) approaches allowing the rapid generation from TNA extracts of FL-cDNAs under the control of the T7 promoter and the successful inoculation of plants using in vitro transcripts obtained from these uncloned amplification products have been developed. We also show that the yeast homologous recombination system permits efficient cloning of FL-cDNAs and the simultaneous one-step tailoring of a ternary Yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector allowing efficient inoculation of both herbaceous and woody host plants by agroinfiltration. Conclusions The fast and efficient strategies described here should have broad applications, in particular for the study "difficult" plant viruses, such as those infecting woody hosts, and potentially for other, non plant-infecting viral agents

Rancang Bangun Sistem Pengukuran Posisi Target dengan Kamera Stereo untuk Pengarah Senjata Otomatis

Salah satu hal yang penting dalam mengarahkan senjata secara otomatis ke target adalah informasi posisi dari target terhadap senjata. Terdapat banyak metode untuk mengetahui posisi target. Salah satunya adalah dengan metode pengukuran triangulasi. Metode ini membutuhkan minimal dua citra untuk medapatkan informasi jarak target terhadap kamera. Kemudian, informasi jarak tersebut bisa diolah untuk mengetahui posisi target terhadap senjata. Di dalam sistem ini, stereo visual digunakan untuk mendukung proses pengukuran triangulasi. Stereo visual menggunakan dua kamera untuk menghasilkan dua citra. Dalam sistem ini, salah satu kamera bertindak sebagai pemilih target. Citra yang ditangkap dua kamera tersebut akan diproses oleh processing unit untuk mendapatkan informasi posisi target terhadap senjata. Informasi ini digunakan untuk menggerakkan motor pada platform senjata agar senjata mengarah ke target. Hasil pengujian yang dilakukan pada sistem ini adalah sistem dapat menentukan posisi target yang dipilih oleh operator dan juga dapat mengarahkan senjata ke arah target tersebut. Akurasi tertinggi dalam penentuan posisi target dicapai ketika jarak antar dua kamera sekitar 30 cm

Guidelines for the reliable use of high throughput sequencing technologies to detect plant pathogens and pests.

High-throughput sequencing (HTS) technologies have the potential to become one of the most signi cant advances in molecular diagnostics. Their use by researchers to detect and characterize plant pathogens and pests has been growing steadily for more than a decade and they are now envisioned as a routine diagnostic test to be deployed by plant pest diagnostics laboratories. Nevertheless, HTS technologies and downstream bioinformatics analysis of the generated datasets represent a complex process including many steps whose reliability must be ensured. The aim of the present guidelines is to provide recommendations for researchers and diagnosticians aiming to reliably use HTS technologies to detect plant pathogens and pests. These guidelines are generic and do not depend on the sequencing technology or platform. They cover all the adoption processes of HTS technologies from test selection to test validation as well as their routine implementation. A special emphasis is given to key elements to be considered: undertaking a risk analysis, designing sample panels for validation, using proper controls, evaluating performance criteria, con rming and interpreting results. These guidelines cover any HTS test used for the detection and identi cation of any plant pest (viroid, virus, bacteria, phytoplasma, fungi and fungus-like protists, nematodes, arthropods, plants) from any type of matrix. Overall, their adoption by diagnosticians and researchers should greatly improve the reliability of pathogens and pest diagnostics and foster the use of HTS technologies in plant health

Statement on a study proposal prepared by the UC to support a future derogation request from the EU import requirements for wood packaging material originating in the US and used to pack and transport military ammunition. Scientific Opinion.

Following a request from the European Commission, the EFSA Panel on Plant Health was asked to deliver a statement on a study proposal prepared by the US to support a future derogation request from the Community import requirements for wood packaging material (WPM) originating in the US and used to pack and transport military ammunition. The Panel reviewed the submitted study proposal and based its conclusions on the shortcomings identified in the study proposal. It was concluded that, due to these shortcomings, the evaluation of the methodological approach described in this study proposal with regard to: 1.the determination of the likelihood that live pinewood nematode (Bursaphelenchus xylophilus) and other harmful organisms relevant for the Community are present in the WPM used by the US Department of Defense to pack and transport military ammunition (Pest survey component), 2. the assessment of the likelihood that harmful organisms may be introduced into the Community through the WPM pathway (Pathway Risk Analysis component) cannot be conducted