High-throughput sequencing (HTS), more specifically RNA sequencing of plant tissues, has become an indispensable tool for plant virologists to detect and identify plant viruses. During the data analysis step, plant virologists typically compare the obtained sequences to reference virus databases. In this way, they are neglecting sequences without homologies to viruses, which usually represent the majority of sequencing reads. We hypothesized that traces of other pathogens might be detected in this unused sequence data. In the present study, our goal was to investigate whether total RNA-seq data, as generated for plant virus detection, is also suitable for the detection of other plant pathogens and pests. As proof of concept, we first analyzed RNA-seq datasets of plant materials with confirmed infections by cellular pathogens in order to check whether these non-viral pathogens could be easily detected in the data. Next, we set up a community effort to re-analyze existing Illumina RNA-seq datasets used for virus detection to check for the potential presence of non-viral pathogens or pests. In total, 101 datasets from 15 participants derived from 51 different plant species were re-analyzed, of which 37 were selected for subsequent in-depth analyses. In 29 of the 37 selected samples (78%), we found convincing traces of non-viral plant pathogens or pests. The organisms most frequently detected in this way were fungi (15/37 datasets), followed by insects (13/37) and mites (9/37). The presence of some of the detected pathogens was confirmed by independent (q)PCRs analyses. After communicating the results, 6 out of the 15 participants indicated that they were unaware of the possible presence of these pathogens in their sample(s). All participants indicated that they would broaden the scope of their bioinformatic analyses in future studies and thus check for the presence of non-viral pathogens. In conclusion, we show that it is possible to detect non-viral pathogens or pests from total RNA-seq datasets, in this case primarily fungi, insects, and mites. With this study, we hope to raise awareness among plant virologists that their data might be useful for fellow plant pathologists in other disciplines (mycology, entomology, bacteriology) as well.

High-throughput sequencing (HTS) is a powerful tool that enables the simultaneous detection and potential identification of any organisms present in a sample. The growing interest in the application of HTS technologies for routine diagnostics in plant health laboratories is triggering the development of guidelines on how to prepare laboratories for performing HTS testing. This paper describes general and technical recommendations to guide laboratories through the complex process of preparing a laboratory for HTS tests within existing quality assurance systems. From nucleic acid extractions to data analysis and interpretation, all of the steps are covered to ensure reliable and reproducible results. These guidelines are relevant for the detection and identification of any plant pest (e.g. arthropods, bacteria, fungi, nematodes, invasive plants or weeds, protozoa, viroids, viruses), and from any type of matrix (e.g. pure microbial culture, plant tissue, soil, water), regardless of the HTS technology (e.g. amplicon sequencing, shotgun sequencing) and of the application (e.g. surveillance programme, phytosanitary certification, quarantine, import control). These guidelines are written in general terms to facilitate the adoption of HTS technologies in plant pest routine diagnostics and enable broader application in all plant health fields, including research. A glossary of relevant terms is provided among the Supplementary Material.

Plant pathogens cause significant reductions in yield and crop quality and cause enormous economic losses worldwide. Reducing these losses provides an obvious strategy to increase food production without further degrading natural ecosystems; however, this requires knowledge of the biology and evolution of the pathogens in agroecosystems.

High-throughput sequencing (HTS) of three pear trees (Pyrus communis) resulted in the identification of two isolates of a Tepovirus species and one isolate of a Robigovirus species, both genera from the family Betaflexiviridae and never reported in pears. Their complete genomes were fully characterized, revealing genomes of about 6.8 kb (Tepovirus) and 8.4 kb (Robigovirus). According to the species demarcation criteria in the family Betaflexiviridae, the tepovirus isolates reported here are divergent isolates of Prunus Virus T (PrVT). The robigovirus isolate had enough difference in pairwise comparison with its closer related species, cherry virus Turkey (CVTR) and it should be considered a new species of this genus, tentatively named as pomes virus Greece (PVGR). A survey was conducted using specific primers designed for each of the viruses and one additional pear accession tested positive to PrVT. Furthermore, both viruses were successfully graft-transmissible to Malus domestica or Malus micromalus, suggesting their potential to be transmitted to Malus species. This study expands the list of viruses infecting pome fruit trees and augments our knowledge on the molecular diversity and host range evolution of betaflexiviruses.

Sugarcane has been grown in the USA since the 1690 s when it was brought to the country by early explorers. New sugarcane varieties and true seed were brought in from other countries, marking the first effort at variety development. A breeding program was established in 1919 to overcome a devastating mosaic virus epidemic and continues to this day. Sugarcane breeding in the USA is conducted by federal, state, and industry programs working together for the domestic industry. Sugarcane is grown in Louisiana, Florida, and Texas. Because of diverse growing environments, separate breeding and selection programs operate in each state, with cooperation among the groups. Louisiana sugarcane production is carried out by over 450 family farming operations, while the production in Florida is carried out by larger corporations. The Texas industry is small, with only a single mill run by a grower’s cooperative. Through decades of successful breeding, sugar recovery has doubled since the 1890s from an average of 5.8–11.7%. A long-term introgression effort has been funded through the USDA at Houma, Louisiana, since the 1950s, and the incorporation of traits from the wild relative, S. spontaneum, is credited with continued yield increases. The USA also maintains the World Collection of Sugarcane and Related Grasses at the USDA-ARS Subtropical Horticulture Research Station, Miami, Florida. This collection serves as a source of germplasm for sugarcane breeders around the world. Sugarcane clones are imported though the USDA-APHIS-Plant Protection and Quarantine Facility in Beltsville, Maryland, where it undergoes extensive disease testing and insect inspection before being released for breeding.

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.

High-throughput sequencing (HTS) technologies have become indispensable tools assisting plant virus diagnostics and research thanks to their ability to detect any plant virus in a sample without prior knowledge. As HTS technologies are heavily relying on bioinformatics analysis of the huge amount of generated sequences, it is of utmost importance that researchers can rely on efficient and reliable bioinformatic tools and can understand the principles, advantages, and disadvantages of the tools used. Here, we present a critical overview of the steps involved in HTS as employed for plant virus detection and virome characterization. We start from sample preparation and nucleic acid extraction as appropriate to the chosen HTS strategy, which is followed by basic data analysis requirements, an extensive overview of the in-depth data processing options, and taxonomic classification of viral sequences detected. By presenting the bioinformatic tools and a detailed overview of the consecutive steps that can be used to implement a well-structured HTS data analysis in an easy and accessible way, this paper is targeted at both beginners and expert scientists engaging in HTS plant virome projects.

Fungi in the genus Clarireedia are widespread and destructive pathogens of grasses worldwide, and are best known as the causal agents of dollar spot disease in turfgrass. Here, we report genome assemblies of seven Clarireedia isolates, including ex-types of the two most widespread species, Clarireedia jacksonii and C. monteithiana. These datasets provide a valuable resource for ongoing studies of the dollar spot pathogens that include population diversity, host-pathogen interactions, marker development, and disease control.

Miscanthus sinensis is a grass used for sugarcane breeding and bioenergy production. Using high throughput sequencing technologies, we identified a new viral genome in infected M. sinensis leaf tissue displaying yellow fleck symptoms. This virus is most related to members of the genus Polerovirus in the family Luteoviridae. The canonical ORFs were computationally identified, the P3 coat protein was expressed, and virus-like particles were purified and found to conform to icosahedral shapes, characteristic of the family Luteoviridae. We propose the name Miscanthus yellow fleck virus for this new virus.

Boxwood blight is a disease threat to natural and managed landscapes worldwide. To determine mating potential of the fungi responsible for the disease, Calonectria pseudonaviculata and C. henricotiae, we characterized their mating-type (MAT) loci. Genomes of C. henricotiae, C. pseudonaviculata and two other Calonectria species (C. leucothoes, C. naviculata) were sequenced and used to design PCR tests for mating-type from 268 isolates collected from four continents. All four Calonectria species have a MAT locus that is structurally consistent with the organization found in heterothallic ascomycetes, with just one idiomorph per individual isolate. Mating type was subdivided by species: all C. henricotiae isolates possessed the MAT1-1 idiomorph, whereas all C. pseudonaviculata isolates possessed the MAT1-2 idiomorph. To determine the potential for divergence at the MAT1 locus to present a barrier to interspecific hybridization, evolutionary analysis was conducted. Phylogenomic estimates showed that C. henricotiae and C. pseudonaviculata diverged approximately 2.1 Mya. However, syntenic comparisons, phylogenetic analyses, and estimates of nucleotide divergence across the MAT1 locus and proximal genes identified minimal divergence in this region of the genome. These results show that in North America and parts of Europe, where only C. pseudonaviculata resides, mating is constrained by the absence of MAT1-1. In regions of Europe where C. henricotiae and C. pseudonaviculata currently share the same host and geographic range, it remains to be determined whether or not these two recently diverged species are able to overcome species barriers to mate.

HomePlant DiseaseVol. 102, No. 2First Report of Barley virus G in Switchgrass (Panicum virgatum) PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Barley virus G in Switchgrass (Panicum virgatum)L. M. Kumar, J. A. Foster, C. McFarland, and M. Malapi-WightL. M. KumarSearch for more papers by this author, J. A. FosterSearch for more papers by this author, C. McFarlandSearch for more papers by this author, and M. Malapi-Wight†Corresponding author: M. Malapi-Wight; E-mail: E-mail Address: martha.malapi-wight@aphis.usda.govSearch for more papers by this authorAffiliationsAuthors and Affiliations L. M. Kumar J. A. Foster , USDA-APHIS-PPQ-Field Operations, Plant Germplasm Quarantine Program, Beltsville, MD 20705 C. McFarland , USDA-APHIS-PPQ-Field Operations, Raleigh, NC 27606 M. Malapi-Wight † , USDA-APHIS-PPQ-Field Operations, Plant Germplasm Quarantine Program, Beltsville, MD 20705. Published Online:30 Nov 2017https://doi.org/10.1094/PDIS-09-17-1390-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Switchgrass (Panicum virgatum L.) is a perennial biobased economic crop used for forage, phytoremediation, as an ornamental, and as a model biomass crop with significant cellulosic biofuel potential. In May 2016, mosaic-like leaf patterns of light and dark green areas were observed on a cultivar Purple Breeze switchgrass clone imported into the USDA-APHIS Plant Germplasm Quarantine Program facilities from the Netherlands. Symptomatic leaf tissue was collected, and total RNA was extracted from pooled leaves using an RNeasy plant mini kit (Qiagen, Valencia, CA). The cDNA library was constructed from ribosomal RNA-depleted extract using the TruSeq Stranded Total RNA with Ribo-Zero plant kit (Illumina, San Diego, CA). Next-generation sequencing (NGS) was performed on an Illumina NextSeq 500 platform, resulting in 112,000,212 single-end reads, 74 bp in length on average, after adapter trimming. Reads were de novo assembled with CLC Genomics Workbench 10.1 software (CLC Bio, Qiagen), of which 275,198 mapped to Barley virus G (BVG; genus Polerovirus; family Luteoviridae). From the 5,620-nucleotide-long consensus sequence, the BVG genome was 100% recovered (GenBank accession no. MF960779) and was 98% identical to the corresponding BVG sequences available in GenBank (KT962089 and LC259081). To confirm the presence of the virus, total RNA was re-extracted from the source plant, and reverse-transcription (RT) PCR was performed using the Luteovirus Group PCR test (Agdia, Elkhart, IN) with a cocktail mix of two primer sets: Luteo-F1/Luteo-F2 and Luteo-R1/Luteo-R2. The resulting 683-bp amplicon was directly sequenced. Pairwise comparisons of the Sanger-sequenced amplicon revealed 97% identity with the BVG sequence derived by NGS from this study, and 96 and 95% identity with BVG-Gimje (KT962089) and BVG-Uiseong (LC259081), respectively. BVG was recently discovered in Korea and has been reported in barley (Hordeum vulgare L.; Zhao et al. 2016), foxtail millet (Setaria italica; Oh et al. 2017), and proso millet (Panicum miliaceum L.; Park et al. 2017). To our knowledge, this is the first report of BVG in P. virgatum. The occurrence of BVG on switchgrass has the dual impact of potentially reducing biomass yield and concomitantly threatening susceptible cereal crops. Further studies are needed to elucidate the distribution, transmission, and host range of BVG.References:Oh, J., et al. 2017. Plant Dis. 101:1061. https://doi.org/10.1094/PDIS-01-17-0036-PDN Link, ISI, Google ScholarPark, C. Y., et al. 2017. Plant Dis. 101:393. https://doi.org/10.1094/PDIS-07-16-0952-PDN Link, ISI, Google ScholarZhao, F., et al. 2016. Arch. Virol. 161:2047. https://doi.org/10.1007/s00705-016-2881-0 Crossref, ISI, Google ScholarDetailsFiguresLiterature CitedRelated Vol. 102, No. 2 February 2018SubscribeISSN:0191-2917e-ISSN:1943-7692 Metrics Article History Issue Date: 19 Jan 2018Published: 30 Nov 2017First Look: 9 Oct 2017Accepted: 5 Oct 2017 Pages: 466-466 InformationThis article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 2018.Cited byBarley virus G (Barley virus G)CABI Compendium, Vol. CABI CompendiumIdentification and characterization of Miscanthus yellow fleck virus, a new polerovirus infecting Miscanthus sinensis17 September 2020 | PLOS ONE, Vol. 15, No. 9Millet Could Be both a Weed and Serve as a Virus Reservoir in Crop Fields28 July 2020 | Plants, Vol. 9, No. 8Maize Lethal Necrosis: An Emerging, Synergistic Viral DiseaseAnnual Review of Virology, Vol. 5, No. 1

Boxwood (Buxus spp.) are broad-leaved, evergreen landscape plants valued for their longevity and ornamental qualities. Volutella leaf and stem blight, caused by the ascomycete fungi Pseudonectria foliicola and P. buxi, is one of the major diseases affecting the health and ornamental qualities of boxwood. Although this disease is less severe than boxwood blight caused by Calonectria pseudonaviculata and C. henricotiae, its widespread occurrence and disfiguring symptoms have caused substantial economic losses to the ornamental industry. In this study, we sequenced the genome of P. foliicola isolate ATCC13545 using Illumina technology and compared it to other publicly available fungal pathogen genomes to better understand the biology of this organism. A de novo assembly estimated the genome size of P. foliicola at 28.7 Mb (425 contigs; N50 = 184,987 bp; avg. coverage 188×), with just 9,272 protein-coding genes. To our knowledge, P. foliicola has the smallest known genome within the Nectriaceae. Consistent with the small size of the genome, the secretome, CAzyme and secondary metabolite profiles of this fungus are reduced relative to two other surveyed Nectriaceae fungal genomes: Dactylonectria macrodidyma JAC15-245 and Fusarium graminearum Ph-1. Interestingly, a large cohort of genes associated with reduced virulence and loss of pathogenicity was identified from the P. foliicola dataset. These data are consistent with the latest observations by plant pathologists that P. buxi and most likely P. foliicola, are opportunistic, latent pathogens that prey upon weak and stressed boxwood plants.

Early and accurate diagnosis of new plant pathogens is vital for the rapid implementation of effective mitigation strategies and appropriate regulatory responses. Most commonly, pathogen identification relies on morphology and DNA marker analysis. However, for new diseases, these approaches may not be sufficient for precise diagnosis. In this study, we used whole-genome sequencing (WGS) to identify the causal agent of a new disease affecting Sarcococca hookeriana (sarcococca). Blight symptoms were observed on sarcococca and adjacent Buxus sempervirens (boxwood) plants in Maryland during 2014. Symptoms on sarcococca were novel, and included twig dieback and dark lesions on leaves and stems. A Calonectria sp. was isolated from both hosts and used to fulfill Koch's postulates but morphology and marker sequence data precluded species-level identification. A 51.4-Mb WGS was generated for the two isolates and identified both as Calonectria pseudonaviculata. A single-nucleotide polymorphism at a noncoding site differentiated between the two host isolates. These results indicate that the same C. pseudonaviculata genotype has the ability to induce disease on both plant species. This study marks the first application of WGS for fungal plant pathogen diagnosis and demonstrates the power of this approach to rapidly identify causal agents of new diseases.

Defining syntenic relationships among orthologous gene clusters is a frequent undertaking of biologists studying organismal evolution through comparative genomic approaches. With the increasing availability of genome data made possible through next-generation sequencing technology, there is a growing need for user-friendly tools capable of assessing synteny. Here we present SimpleSynteny, a new web-based platform capable of directly interrogating collinearity of local genomic neighbors across multiple species in a targeted manner. SimpleSynteny provides a pipeline for evaluating the synteny of a preselected set of gene targets across multiple organismal genomes. An emphasis has been placed on ease-of-use, and users are only required to submit FASTA files for their genomes and genes of interest. SimpleSynteny then guides the user through an iterative process of exploring and customizing genomes individually before combining them into a final high-resolution figure. Because the process is iterative, it allows the user to customize the organization of multiple contigs and incorporate knowledge from additional sources, rather than forcing complete dependence on the computational predictions. Additional tools are provided to help the user identify which contigs in a genome assembly contain gene targets and to optimize analyses of circular genomes. SimpleSynteny is freely available at: http://www.SimpleSynteny.com.

Rapid and accurate molecular diagnostic tools are critical to efforts to minimize the impact and spread of emergent pathogens. The identification of diagnostic markers for novel pathogens presents several challenges, especially in the absence of information about population diversity and where genetic resources are limited. The objective of this study was to use comparative genomics datasets to find unique target regions suitable for the diagnosis of two fungal species causing a newly emergent blight disease of boxwood. Candidate marker regions for loop-mediated isothermal amplification (LAMP) assays were identified from draft genomes of Calonectria henricotiae and C. pseudonaviculata, as well as three related species not associated with this disease. To increase the probability of identifying unique targets, we used three approaches to mine genome datasets, based on (i) unique regions, (ii) polymorphisms, and (iii) presence/absence of regions across datasets. From a pool of candidate markers, we demonstrate LAMP assay specificity by testing related fungal species, common boxwood pathogens, and environmental samples containing 445 diverse fungal taxa. This comparative-genomics-based approach to the development of LAMP diagnostic assays is the first of its kind for fungi and could be easily applied to diagnostic marker development for other newly emergent plant pathogens.

ABSTRACT Dactylonectria macrodidyma is part of the Nectriaceae , a family containing important plant pathogens. This species possesses the ability to induce disease on grapevine, avocado, and olive. Here, we report the first draft genome of D. macrodidyma isolate JAC15-245. The assembled genome was 58 Mbp and contained an estimated 16,454 genes.

The genomes of Chrysoporthe austroafricana, Diplodia scrobiculata, Fusarium nygami, Leptographium lundbergii, Limonomyces culmigenus, Stagonosporopsis tanaceti, and Thielaviopsis punctulata are presented in this genome announcement. These seven genomes are from endophytes, plant pathogens and economically important fungal species. The genome sizes range from 26.6 Mb in the case of Leptographium lundbergii to 44 Mb for Chrysoporthe austroafricana. The availability of these genome data will provide opportunities to resolve longstanding questions regarding the taxonomy of species in these genera, and may contribute to our understanding of the lifestyles through comparative studies with closely related organisms.

Premise of the study: Genic microsatellites or simple sequence repeat (genic-SSR) markers were developed in boxwood (Buxus taxa) for genetic diversity analysis, identification of taxa, and to facilitate breeding.Methods and Results: cDNA libraries were developed from mRNA extracted from leaves of Buxus sempervirens 'Vardar Valley' and sequenced using the Illumina MiSeq system. Approximately 11.9 million base pairs of sequence data were examined and 845 genic-SSRs were identified, including 469 dinucleotide, 360 trinucleotide, seven tetranucleotide, one pentanucleotide, and eight hexanucleotide repeats. Primer pairs were designed for 71 selectively chosen genic-SSRs containing trinucleotide repeat motifs and were used to amplify the corresponding loci in 18 diverse boxwood accessions. Twenty-three primer pairs amplified polymorphic loci, with two to 10 alleles per locus.Conclusions: These novel polymorphic genic-SSR markers will aid in evaluating genetic diversity of boxwood germplasm and allow verification of hybrids and cultivars for breeding programs.