Tomato is an important vegetable in the United States and around the world. Recently, tomato brown rugose fruit virus (ToBRFV), an emerging tobamovirus, has impacted tomato crops worldwide and can result in fruit loss. ToBRFV causes severe symptoms, such as mosaic, puckering, and necrotic lesions on leaves; other symptoms include brown rugose and marbling on fruits. More importantly, ToBRFV can overcome resistance in tomato cultivars carrying the Tm-2² locus. In this study, we recovered ToBRFV sequences from tomato seeds, leaves, and fruits from the U.S., Mexico, and Peru. Samples were pre-screened using a real-time RT-PCR assay prior to high-throughput sequencing. Virus draft genomes from 22 samples were assembled and analyzed against more than 120 publicly available genomes. Overall, most sequenced isolates were similar to each other and did not form a distinct population. Phylogenetic analysis revealed three clades within the ToBRFV population. Most of the isolates (95%) clustered in clade 3. Genetic analysis revealed differentiation between the three clades indicating minor divergence occurring. Overall, pairwise identity showed limited genetic diversity among the isolates in this study with worldwide isolates, with a pairwise identity ranging from 99.36% and 99.97%. The overall population is undergoing high gene flow and population expansion with strong negative selection pressure at all ToBRFV genes. Based on the results of this study, it is likely that the limited ToBRFV diversity is associated with the rapid movement and eradication of ToBRFV-infected material between countries.

Next-generation sequencing technologies have aided molecular detection and diagnosis of plant pathogens. Short read sequencing technologies can be expensive, time consuming, and lack portability. The Oxford Nanopore Technologies (ONT) MinION sequencer offers plant diagnosticians an alternative for whole-genome and transcriptome sequencing with the advantages of portability, rapid results and access to sequences generated, and relatively low cost. Although this company offers multiple sequencing products, most previous articles focused on comparing this technology with other sequencing technology (Sanger, Illumina) and not comparing ONT product performance. In this project, we explored the potential of using ONT’s kits without modifications for the sequencing of plant pathogens. We compared the performance of DNA library preparation protocols (1D versus 1D² versus Rapid) and RNA library preparation protocols (cDNA PCR Sequencing versus Direct RNA) on the MinION sequencer and examined different data analysis approaches for two well-characterized plant pathogens of agricultural concern, Ralstonia solanacearum and plum pox virus. For bacterial DNA sequencing, the Rapid Sequencing kit had the lowest throughput but offered the shortest sample-to-sequence turnaround time. The cDNA PCR Sequencing kit was better suited for plant RNA viruses than the Direct RNA. Whole-genome assemblies using all tested kits provided accurate identification of the target pathogen when pure cultures or infected plant material was used. This technology offers a shorter sample-to-sequence timeline than previous technologies for both DNA and RNA samples. Data analysis can still be time- and resource-consuming, but we found that 1 h of data generation was sufficient for accurate identification of the target pathogens, which can alleviate computing resources because a full 48-h run of sequencing data is not required. With the decreasing costs of flow cells, MinION is now an additional tool to be considered by plant diagnosticians for whole-genome and transcriptome-based diagnostics.

Citrus yellow vein clearing virus is a previously reported citrus virus from Asia with widespread distribution in China. In 2022, the California Department of Food and Agriculture conducted a multipest citrus survey targeting multiple citrus pathogens including citrus yellow vein clearing virus (CYVCV). In March 2022, a lemon tree with symptoms of vein clearing, chlorosis, and mottling in a private garden in the city of Tulare, California, tested positive for CYVCV, which triggered an intensive survey in the surrounding areas. A total of 3,019 plant samples, including citrus and noncitrus species, were collected and tested for CYVCV using conventional reverse transcription polymerase chain reaction, reverse transcription quantitative polymerase chain reaction, and Sanger sequencing. Five hundred eighty-six citrus trees tested positive for CYVCV, including eight citrus species not previously recorded infected under field conditions. Comparative genomic studies were conducted using 17 complete viral genomes. Sequence analysis revealed two major phylogenetic groups. Known Asian isolates and five California isolates from this study made up the first group, whereas all other CYVCV isolates from California formed a second group, distinct from all worldwide isolates. Overall, the CYVCV population shows rapid expansion and high differentiation indicating a population bottleneck typical of a recent introduction into a new geographic area.

Passiflora edulis, commonly known as passion fruit, is a vine species of passionflower native to South America. In Colombia, yellow passion fruit (P. edulis f. flavicarpa) is the most important spe...