Ash shoestring-associated virus (ASaV; Emaravirus fraxini) is a five-segmented, negative-sense RNA virus associated with chlorosis and leaf deformation in Fraxinus species. This study investigated the genetic diversity and evolutionary dynamics of ASaV by analyzing nearly full-length RNA2–RNA5 and partial RNA1 sequences from isolates collected from different geographic regions and Fraxinus hosts. The sequence data uncovered that ASaV has a conserved genome, in which RNA3 and RNA5 showed more genetic divergence than other segments in the sequenced isolates. ASaV RNA3 and partial RNA1 were the most informative genomic regions for phylogenetic studies. There was a correlation between the clustering of the ASaV isolates and host species when the phylogenetic tree was constructed based on the RNA1 region. The ASaV genome is predominantly under purifying selection. Newly designed primers in this study facilitated robust amplification of genomic regions.

This review describes the development of the bioassay as a means of quantifying plant viruses, with particular attention to tobamovirus. It delves into various models used to establish a correlation between virus particle concentration and the number of induced local lesions (the infectivity dilution curve), including the Poisson, Furumoto and Mickey, Kleczkowski, Growth curve, and modified Poisson models. The parameters of each model are described, and their application or performance in the context of the tobacco mosaic virus is explored. This overview highlights the enduring value of the infectivity dilution curve in tobamovirus quantification, providing valuable insights for researchers or practitioners of bioassays and theoreticians of modeling.

Introduction: Viruses are increasingly recognized as integral components of plant-associated biological systems. However, their occurrence and diversity in the reproductive tissues of woody plants remain poorly understood. Birch ( Betula spp.) produces large quantities of wind-dispersed pollen that can travel over long distances and may harbour viruses or virus-derived nucleic acids originating from the host plant and/or its associated microbiota. Methods: We investigated the virome of birch pollen collected from trees growing in central and suburban Berlin, Germany. Metatranscriptomic analyses were performed on pooled pollen samples collected in 2020. These analyses were complemented by RT-PCR screening of individually processed pollen samples collected in 2025 from resampled trees. Primer walking was additionally used to recover an extended genome sequence of a pollen-associated birch toti-like virus. Results: Multiple virus-associated contigs were identified in both pooled metatranscriptomic datasets. These included sequences corresponding to the cherry leaf roll virus (CLRV), the birch idaeovirus (BIV) and the birch toti-like virus (BTLV), as well as additional virus-like contigs provisionally assigned to lineages related to the Orthototiviridae, Botourmiaviridae, Endornaviridae , and Chrysoviridae families. RT-PCR analysis of individually processed pollen samples confirmed the continued detection of CLRV, BIV, and BTLV within the Berlin sampling framework. A near-complete genome sequence was recovered from a pollen-derived BTLV isolate from Berlin, showing high amino acid sequence identity to a recently described leaf-derived BTLV isolate from the United States. Discussion: These findings demonstrate that birch pollen harbours a diverse assemblage of plant- and/or microbiome-associated viruses and expand the known tissue distribution and geographic range of BTLV. More broadly, they establish pollen as an underexplored ecological niche for virome research and provide a foundation for future studies on the ecology, transmission dynamics, and epidemiological significance of pollen-associated and pollen-transmitted viruses.