PhytoPipe: a phytosanitary pipeline for plant pathogen detection and diagnosis using RNA-seq data.

BACKGROUND: Detection of exotic plant pathogens and preventing their entry and establishment are critical for the protection of agricultural systems while securing the global trading of agricultural commodities. High-throughput sequencing (HTS) has been applied successfully for plant pathogen discovery, leading to its current application in routine pathogen detection. However, the analysis of massive amounts of HTS data has become one of the major challenges for the use of HTS more broadly as a rapid diagnostics tool. Several bioinformatics pipelines have been developed to handle HTS data with a focus on plant virus and viroid detection. However, there is a need for an integrative tool that can simultaneously detect a wider range of other plant pathogens in HTS data, such as bacteria (including phytoplasmas), fungi, and oomycetes, and this tool should also be capable of generating a comprehensive report on the phytosanitary status of the diagnosed specimen. RESULTS: We have developed an open-source bioinformatics pipeline called PhytoPipe (Phytosanitary Pipeline) to provide the plant pathology diagnostician community with a user-friendly tool that integrates analysis and visualization of HTS RNA-seq data. PhytoPipe includes quality control of reads, read classification, assembly-based annotation, and reference-based mapping. The final product of the analysis is a comprehensive report for easy interpretation of not only viruses and viroids but also bacteria (including phytoplasma), fungi, and oomycetes. PhytoPipe is implemented in Snakemake workflow with Python 3 and bash scripts in a Linux environment. The source code for PhytoPipe is freely available and distributed under a BSD-3 license. CONCLUSIONS: PhytoPipe provides an integrative bioinformatics pipeline that can be used for the analysis of HTS RNA-seq data. PhytoPipe is easily installed on a Linux or Mac system and can be conveniently used with a Docker image, which includes all dependent packages and software related to analyses. It is publicly available on GitHub at https://github.com/healthyPlant/PhytoPipe and on Docker Hub at https://hub.docker.com/r/healthyplant/phytopipe .

Complete genome sequence of zoysia mosaic virus, a novel member of the genus Poacevirus.

Here, we report the detection and characterization of the genome of a novel poacevirus isolated from Zoysia matrella (Merrill) imported into the United States from Japan. The novel virus, tentatively named "zoysia mosaic virus" (ZoMV), is a single-stranded RNA virus with a genome of 9,728 nucleotides (nt) in length, encoding a large putative polyprotein of 3,119 amino acids (aa). The ZoMV genome is closely related to the triticum mosaic virus (TriMV; FJ263671) genome, with 57.18% nt and 51.74% aa sequence identity in the polyprotein region. Moreover, phylogenetic analysis showed that ZoMV is closely related to all other members of the genus Poacevirus. A survey of imported grasses showed that ZoMV was detected only in zoysiagrass. This is the first report of the complete genome sequence of a novel viral pathogen of zoysiagrass of the genus Poacevirus, for which we propose the binomial species name "Poacevirus zoisiae".

Molecular characterization of horse nettle virus A, a new member of subgroup B of the genus Nepovirus.

A new positive-strand RNA virus was discovered in a horse nettle plant, using high-throughput sequencing (HTS), and its complete genome, consisting of RNA1 and RNA2, which are 7522 and 4710 nucleotides in length, respectively, was characterized. Each genome segment contains a single open reading frame flanked by 5' and 3' untranslated regions (UTRs), followed by a poly(A) tail at the 3' end. The encoded proteins have the highest amino acid sequence identity (55% and 45%) to the polyprotein encoded by RNA1 of tomato black ring virus (TBRV) and RNA2 of potato virus B (PVB), respectively. Its genome organization and phylogenetic relationship to other nepoviruses suggested that this virus is a novel member of subgroup B, and recombination analysis revealed its evolutionary history within the subgroup. These results suggest the new virus, provisionally named "horse nettle virus A", represents a new species within the genus Nepovirus.

High-throughput detection of a large set of viruses and viroids of pome and stone fruit trees by multiplex PCR-based amplicon sequencing.

A comprehensive diagnostic method of known plant viruses and viroids is necessary to provide an accurate phytosanitary status of fruit trees. However, most widely used detection methods have a small limit on either the number of targeted viruses/viroids or the number of samples to be evaluated at a time, hampering the ability to rapidly scale up the test capacity. Here we report that by combining the power of high multiplexing PCR (499 primer pairs) of small amplicons (120-135bp), targeting 27 viruses and 7 viroids of fruit trees, followed by a single high-throughput sequencing (HTS) run, we accurately diagnosed the viruses and viroids on as many as 123 pome and stone fruit tree samples. We compared the accuracy, sensitivity, and reproducibility of this approach and contrast it with other detection methods including HTS of total RNA (RNA-Seq) and individual RT-qPCR for every fruit tree virus or viroid under the study. We argue that this robust and high-throughput cost-effective diagnostic tool will enhance the viral/viroid knowledge of fruit trees while increasing the capacity for large scale diagnostics. This approach can also be adopted for the detection of multiple viruses and viroids in other crops.

HTS-Based Diagnostics of Sugarcane Viruses: Seasonal Variation and Its Implications for Accurate Detection.

Rapid global germplasm trade has increased concern about the spread of plant pathogens and pests across borders that could become established, affecting agriculture and environment systems. Viral pathogens are of particular concern due to their difficulty to control once established. A comprehensive diagnostic platform that accurately detects both known and unknown virus species, as well as unreported variants, is playing a pivotal role across plant germplasm quarantine programs. Here we propose the addition of high-throughput sequencing (HTS) from total RNA to the routine quarantine diagnostic workflow of sugarcane viruses. We evaluated the impact of sequencing depth needed for the HTS-based identification of seven regulated sugarcane RNA/DNA viruses across two different growing seasons (spring and fall). Our HTS analysis revealed that viral normalized read counts (RPKM) was up to 23-times higher in spring than in the fall season for six out of the seven viruses. Random read subsampling analyses suggested that the minimum number of reads required for reliable detection of RNA viruses was 0.5 million, with a viral genome coverage of at least 92%. Using an HTS-based total RNA metagenomics approach, we identified all targeted viruses independent of the time of the year, highlighting that higher sequencing depth is needed for the identification of DNA viruses.

HHS/CDC legal response to SARS outbreak.

Before the severe acute respiratory syndrome (SARS) outbreak, the Centers for Disease Control and Prevention's (CDC) legal authority to apprehend, detain, or conditionally release persons was limited to seven listed diseases, not including SARS, and could only be changed using a two-step process: 1) executive order of the President of the United States on recommendation by the Secretary, U.S. Department of Health and Human Services (HHS), and 2) amendment to CDC quarantine regulations (42 CFR Parts 70 and 71). In April 2003, in response to the SARS outbreak, the federal executive branch acted rapidly to add SARS to the list of quarantinable communicable diseases. At the same time, HHS amended the regulations to streamline the process of adding future emerging infectious diseases. Since the emergence of SARS, CDC has increased legal preparedness for future public health emergencies by establishing a multistate teleconference program for public health lawyers and a Web-based clearinghouse of legal documents.