du Toit, L. J. | bioRxiv Science

bioRxiv2026

Deep sequencing of High Plains wheat mosaic virus from sweet corn to guide seed health testing reveals multiple variants for all eight genome segments and two major isolate types

High Plains wheat mosaic virus (HPWMoV) is a wheat and maize-infecting virus of phytosanitary concern due to its potential for seed transmission. Recent phytosanitary restrictions have required sweet corn seed lots to test negative for HPWMoV prior to import into certain countries. To inform the design of more sensitive and broad-spectrum diagnostic primers for seed health testing and phytosanitary certification, we performed deep sequencing of HPWMoV-positive tissue collected from fields in two major sweet corn seed production regions in the Pacific Northwest, the Columbia Basin and Treasure Valley. Virus-like particle enrichment prior to Illumina sequencing facilitated near complete genome coverage (>95%) for the 21 HPWMoV isolates sequenced. De novo assembly of the eight viral genome segments revealed high levels of diversity for each segment, with at least two variants identified for each RNA and three variants for RNA3, RNA6, and RNA8. Within each sample, only one variant per RNA segment was usually present, with the notable exception of RNA3, sorting each isolate into what we designated type A and type B isolates. All but one previously sequenced HPWMoV isolate can be sorted into these two types. Two samples contained at least two variants for every RNA, totaling 17 genome segments, potentially representing a co-infection of type A and type B isolates. Despite this variability, we successfully designed two primer and probe sets for reverse transcription-quantitative polymerase chain reactions (RT-qPCR) that detected all 20 isolates tested in a duplex diagnostic assay, making the assay suitable for seed health testing for HPWMoV.

plant biology↗

bioRxiv2024

Plasmids encode and can mobilize onion pathogenicity in Pantoea agglomerans

Pantoea agglomerans is one of four Pantoea species for which strains have been reported in the United States to cause bacterial rot of onion bulbs. However, not all P. agglomerans strains are pathogenic to onion. We characterized onion-associated strains of P. agglomerans to elucidate the genetic and genomic signatures of onion-pathogenic P. agglomerans. We collected >300 P. agglomerans strains associated with symptomatic onion plants and bulbs from public culture collections, research laboratories, and a multi-year survey in 11 states in the USA. Genome assemblies were generated for 87 P. agglomerans strains that showed a range in onion virulence phenotypes. Combining the 87 genome assemblies with 100 high-quality, public P. agglomerans genome assemblies identified two well-represented and well-supported P. agglomerans phylogroups. Strains causing severe symptoms on onion leaves and bulbs were only identified in Phylogroup II and encoded the HiVir biosynthetic cluster for the phytotoxin pantaphos, supporting the role of HiVir as a crucial pathogenicity factor. Using a MASH-based plasmid classification system, the P. agglomerans HiVir cluster was determined to be encoded in two distinct plasmid contexts: 1) as an accessory gene cluster on a conserved P. agglomerans plasmid (pAggl), or 2) on a mosaic cluster of plasmids common among onion strains (pOnion). Analysis of closed genomes of P. agglomerans revealed that the pOnion plasmids harbored alt genes responsible for encoding tolerance to the thiosulfinate defensive chemistry in Allium spp. Additionally, many of these pOnion plasmids harbored cop gene clusters, which confer resistance to copper. However, the pOnion plasmids encoded the HiVir cluster less frequently. We demonstrated that the pOnion plasmid pCB1C, encoding HiVir and alt clusters as well as an intact conjugative type IV secretion system (T4SS), can act as a natively mobilizable pathogenicity plasmid that transforms P. agglomerans Phylogroup I strains, including environmental strains, into virulent pathogens of onion. This work indicates a central role for plasmids and plasmid ecology in mediating P. agglomerans interactions with onion plants, with potential implications for onion bacterial disease management.

microbiology↗