High-throughput sequencing (HTS) technologies have revolutionized plant virology through simultaneous detection of mixed viral infections. HTS advances have uncovered and improved understanding of virus biology, ecology, and evolution which is vital for viral disease management. Plant viruses continue to threaten global agricultural productivity and strict quarantine measures are essential to prevent the introduction and spread of virulent viruses around the world. The gradual decrease in HTS operational costs, including improved computational systems and automation through robotics, has facilitated the adoption of this tool for plant diagnostics, including its use in surveillance and quarantine programs. However, the speed of technology advancements and distinct HTS chemistries, laboratory procedures, data management, and bioinformatic analyses have proven challenging. In addition, the lack of viral species reference sequences, compared with the estimated number of distinct viral taxa, makes classification and identification of novel viruses difficult. There is a need for standardized HTS testing, especially within plant health programs. In this review, we consider the application of HTS in plant virology, explore the technical challenges faced and the opportunities for HTS in plant health certification. We propose standards for overcoming current barriers and for ensuring reliable and reproducible results. These efforts will impact global plant health by reducing the risk of introduction and the spread of damaging novel viruses.

The African eggplant (Solanum aethiopicum) is a nutritious traditional vegetable used in many African countries, including Uganda and Nigeria. It is thought to have been domesticated in Africa from its wild relative, Solanum anguivi. S. aethiopicum has been routinely used as a source of disease resistance genes for several Solanaceae crops, including Solanum melongena. A lack of genomic resources has meant that breeding of S. aethiopicum has lagged behind other vegetable crops. We assembled a 1.02-Gb draft genome of S. aethiopicum, which contained predominantly repetitive sequences (78.9%). We annotated 37,681 gene models, including 34,906 protein-coding genes. Expansion of disease resistance genes was observed via 2 rounds of amplification of long terminal repeat retrotransposons, which may have occurred ∼1.25 and 3.5 million years ago, respectively. By resequencing 65 S. aethiopicum and S. anguivi genotypes, 18,614,838 single-nucleotide polymorphisms were identified, of which 34,171 were located within disease resistance genes. Analysis of domestication and demographic history revealed active selection for genes involved in drought tolerance in both “Gilo” and “Shum” groups. A pan-genome of S. aethiopicum was assembled, containing 51,351 protein-coding genes; 7,069 of these genes were missing from the reference genome. The genome sequence of S. aethiopicum enhances our understanding of its biotic and abiotic resistance. The single-nucleotide polymorphisms identified are immediately available for use by breeders. The information provided here will accelerate selection and breeding of the African eggplant, as well as other crops within the Solanaceae family.

Australia is a major grain exporter, and this trade makes an important contribution to its economy. Fortunately, it remains free of many damaging virus diseases and virus vectors found elsewhere. However, its crop biosecurity is under increasing pressure from global ecological, climatic, and demographic challenges. Stringent biosecurity and plant health programs safeguard Australian grain production from damaging virus and virus vector incursions entering via different pathways. These programs formerly relied upon traditional testing procedures (indicator hosts, serology, PCRs) to intercept incoming virus-contaminated plant material. Recently, the integration of rapid genomic diagnostics innovation involving High Throughput Sequencing (HTS) smart tools into sample testing schedules is under exploration to improve virus testing accuracy, efficiency, and cost effectiveness under diverse circumstances. This process includes evaluating deployment of Illumina and Oxford Nanopore Technology shotgun sequencing. It also includes evaluating targeted viral genome HTS and virus vector metabarcoding approaches. In addition, using machine learning and deep learning capacities for big data analyses and remote sensing technologies will improve virus surveillance. Tracking damaging virus variants will be improved by surveillance networks which combine virus genomic-surveillance systems with an interoperable virus database. Sequencing Australian virus specimen collections will help ensure the accuracy of virus identifications based solely on genetic information. Enhancing routine diagnosis and data collection using these innovations will improve post entry virus interception and background virus and vector surveillance. This will help reduce the frequency of new incursions, improve virus management during eradication, containment and other plant health activities, and achieve more profitable Australian grain production.

High-throughput sequencing (HTS) is an important tool for plant virus detection and discovery. HTS technologies such as Nanopore sequencing has been rapidly developing in recent years, and offers new possibilities for fast routine diagnostic applications. This study compared MiSeq (Illumina) RNA-Seq with the MinION sequencer (Oxford Nanopore Technologies) (ONT) direct-RNA-Seq methods to detect and sequence the broad bean wilt virus 2 (BBWV2) genome. The Illumina BBWV2 RNA1 and RNA2 genome segments were intact and matched with ONT with 99.1% and 98.8% nucleotide identity (nt) match, respectively. The RNA1 genome derived from ONT had deletions within the nucleoside-triphosphate binding and protease factor open-reading frames, and the upstream of five untranslated regions. However, its RNA2 was intact, and no sequencing errors were observed. The ONT and Illumina RNA1 and RNA 2 BBWV2 genome segments clustered together phylogenetically along with other none- Fabaceae species, BBWV2 RNA1 NCBI accession KC790225 , and RNA 2 MW556592 both from China. This is the first BBWV2 genome study reported in Australia and forms part of the strategy to integrate versatile diagnostic genomics tools at the border to safeguard the Australian grains industry. IMPORTANCE: Globally, viral diseases impair the growth and vigor of cultivated crops such as grains, leading to a significant reduction in quality, marketability, and competitiveness. As an island nation, Australia has a distinct advantage in using its border to prevent the introduction of damaging viruses, which threaten the continental agricultural sector. However, breeding programs in Australia rely on imported seeds as new sources of genetic diversity. As such, it is critical to remain vigilant in identifying new and emerging viral pathogens, by ensuring the availability of accurate genomic diagnostic tools at the grain biosecurity border. High-throughput sequencing offers game-changing opportunities in biosecurity routine testing. Genomic results are more accurate and informative compared to traditional molecular methods or biological indexing. The present work contributes to strengthening accurate phytosanitary screening, to safeguard the Australian grains industry, and expedite germplasm release to the end users.

Passion fruit viral diseases pose a significant threat to Kenya’s passion fruit industry. To unravel the complexity of these diseases, comprehensive virus surveys were conducted across major passion fruit-growing counties. Passion fruit woodiness disease symptoms, like fruit hardening, chlorotic mottling, and leaf distortion, were prevalent. The study unveiled the first 23 complete genomes of Ugandan passiflora virus (UPV) and two East Asian passiflora distortion virus (EAPDV) in Kenya. UPV showed 99% nucleotide (nt) match to a UPV genome from Uganda and 66% nt identity match to EAPDV. In addition, UPV variants and two partial passion fruit green spot virus sequences and partial (passiflora emaravirus) segment RNA1-5 (novel allexivirus and an emaravirus, respectively) were detected. Phylogenetic analysis revealed distinct lineages (I–III), indicating potential multiple introductions into Kenya. Recombination analysis detected no significant breakpoints. However, the study proposed the renaming of EAPDV to passiflora distortion virus (PDV) and UPV to passiflora virus (PV) for neutral nomenclature, without geographical association. Additionally, the study highlighted the role of coinfections in symptom expression, suggesting a potential synergistic relationship between PV, PDV, and other viruses. The results recommend stringent management strategies and enhanced surveillance to mitigate the economic impact of these viruses on the Kenyan passion fruit industry. The findings from this study underscore the need to strengthen nursery certification programs and pest diagnostic protocols in Kenya. Additionally, enhanced pest surveillance and import regulations are critical to preventing the introduction and spread of emerging plant viral diseases, thereby safeguarding the country’s horticultural productivity and biosecurity. To our knowledge, this is the first comprehensive study of viral diseases of passion fruit in Kenya. IMPORTANCE: This study presents the first comprehensive survey of viral pathogens affecting passion fruit in Kenya, identifying Ugandan passiflora virus (UPV) and East Asian passiflora distortion virus (EAPDV) as major contributors. Through genomic sequencing, 23 complete genomes of UPV and two of EAPDV were characterized, revealing a 99% nucleotide (nt) similarity between UPV strains from Uganda and Kenya, and 66% nt match with EAPDV. Phylogenetic analysis identified distinct lineages, suggesting possible multiple viral introductions in Kenya. The study also highlights potential synergistic coinfections between UPV, EAPDV, and other viruses, leading to more severe disease symptoms. In light of these findings, the study proposes renaming EAPDV as passiflora distortion virus and UPV as passiflora virus for a more neutral name classification. The research underscores the urgent need for enhanced surveillance, stringent phytosanitary measures, and improved management strategies to mitigate the threat of viral diseases, to safeguard the Kenyan passion fruit industry, and elsewhere.

A Broad bean mottle virus (BBMV) isolate (S52) obtained from an infected Vicia faba leaf sample from Syria was sequenced using Oxford Nanopore long-read sequencing at the Australian border. The genome had 95.6%, 98.2%, and 93.4% nucleotide sequence identity to BBMV strains RNA1 (Bawden), RNA2 (Mo), and RNA3 (Bawden).