Revisiting a pollen-transmitted ilarvirus previously associated with angular mosaic of grapevine.

We report the characterization of a novel tri-segmented RNA virus infecting Mercurialis annua, a common crop weed and model species in plant science. The virus, named "Mercurialis latent virus" (MeLaV) was first identified in a mixed infection with the recently described Mercurialis orthotospovirus 1 (MerV1) on symptomatic plants grown in glasshouses in Lausanne (Switzerland). Both viruses were found to be transmitted by Thrips tabaci, which presumably help the inoculation of infected pollen in the case of MeLaV. Complete genome sequencing of the latter revealed a typical ilarviral architecture and close phylogenetic relationship with members of the Ilarvirus subgroup 1. Surprisingly, a short portion of MeLaV replicase was found to be identical to the partial sequence of grapevine angular mosaic virus (GAMV) reported in Greece in the early 1990s. However, we have compiled data that challenge the involvement of GAMV in angular mosaic of grapevine, and we propose alternative causal agents for this disorder. In parallel, three highly-conserved MeLaV isolates were identified in symptomatic leaf samples in The Netherlands, including a herbarium sample collected in 1991. The virus was also traced in diverse RNA sequencing datasets from 2013 to 2020, corresponding to transcriptomic analyses of M. annua and other plant species from five European countries, as well as metaviromics analyses of bees in Belgium. Additional hosts are thus expected for MeLaV, yet we argue that infected pollen grains have likely contaminated several sequencing datasets and may have caused the initial characterization of MeLaV as GAMV.

Role of Thrips palmi and Parthenium hysterophorus pollen in active spread of tobacco streak virus in the cotton ecosystem.

Tobacco streak virus incidence in the cotton field, cv.CO14 at Department of Cotton, Tamil Nadu Agricultural University (TNAU), Coimbatore, India was nearly 36.50 %. Cotton plants infected with TSV exhibits different types of symptoms, including necrotic spots, lesions, mosaic, purplish necrotic rings, square drying, veinal necrosis and drying of terminal shoots. The highly prevalent thrips species in this cotton ecosystem was established as Thrips palmi (60.00 %) by morphological (ESEM) and molecular methods (RT-PCR using mtCOI primers). The density of the alternate weed host, Parthenium hysterophorus, was 15.05 plants per m2 in these fields. Association of Thrips palmi with Parthenium was confirmed, when observed under environmental scanning electron microscope (ESEM), Parthenium pollen grains (i.e., average size @ 15000X =12.94 µm) were found adhering to its body. Molecular studies through RT-PCR confirmed the presence of TSV in the leaves and pollen grains of symptomatic and symptom-free Parthenium plants collected from the cotton field (cv. CO14). Therefore, the combined role of Thrips palmi and the Parthenium pollen grains in the transmission of TSV was examined; acquiring of TSV and its presence in the body of Thrips palmi instars and adults after 72 h of AAP was convincingly demonstrated using RT-PCR, NASH and qPCR. However virus acquired thrips could not transmit the virus. Pollen from TSV infected Parthenium plants when dusted on cotton (ANKUR 2110) seedlings along with virus acquired or non-acquired thrips led to symptom development 22 days after sowing. From the study it is evident that thrips only facilitate the movement of TSV borne pollen grains, and thereby contributing to active spread of the virus.

Characterization of distinct strains of an aphid-transmitted ilarvirus (Fam. Bromoviridae) infecting different hosts from South America.

Potato yellowing virus (PYV, original code SB-22), an unassigned member of the Genus Ilarvirus Family Bromoviridae, has been reported infecting potatoes in Peru, Ecuador and Chile. It is associated with symptomless infections, however yellowing of young leaves has been observed in some potato cultivars. Thirteen potato and yacon isolates were selected after routine screening of CIP-germplasm and twenty-four were identified from 994 potato plants collected in Peru whereas one was intercepted from yacon in the UK. These isolates were identified using high throughput sequencing, ELISA, host range and RT-PCR. Here we report the sequence characterization of the complete genomes of nine PYV isolates found infecting Solanum tuberosum, four complete genome isolates infecting Smallanthus sonchifolius (yacon), and in addition 15 complete RNA3 sequences from potato and partial sequences of RNA1, 2 and 3 of isolates infecting potato and yacon from Ecuador, Peru and Bolivia. Results of phylogenetic and recombination analysis showed RNA3 to be the most variable among the virus isolates and suggest potato infecting isolates have resulted through acquisition of a movement protein variant through recombination with an unknown but related ilarvirus, whereas one yacon isolate from Bolivia also had resulted from a recombination event with another related viruses in the same region. Yacon isolates could be distinguished from potato isolates by their inability to infect Physalis floridana, and potato isolates from Ecuador and Peru could be distinguished by their symptomatology in this host as well as phylogenetically. The non-recombinant yacon isolates were closely related to a recently described isolate from Solanum muricatum (pepino dulce), and all isolates were related to Fragaria chiloensis latent virus (FCiLV) reported in strawberry from Chile, and probably should be considered the same species. Although PYV is not serologically related to Alfalfa mosaic virus (AMV), they are both transmitted by aphids and share several other characteristics that support the previous suggestion to reclassify AMV as a member in the genus Ilarvirus.

Detection and quantification of four viruses in Prunus pollen: Implications for biosecurity.

Pollen transmitted viruses require accurate detection and identification to minimize the risk of spread through the global import and export of pollen. Therefore in this study we developed RT-qPCR assays for the detection of Cherry leaf roll virus (CLRV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), and Cherry virus A (CVA), four viruses that infect pollen of Prunus species. Assays were designed against alignments of extant sequences, optimized, and specificity was tested against known positive, negative, and non-target controls. An examination of assay sensitivity showed that detection of virus at concentrations as low as 101 copies was possible, although 102 copies was more consistent. Furthermore, comparison against extant assays showed that in both pollen and plant samples, the newly developed RT-qPCR assays were more sensitive and could detect a greater range of isolates than extant endpoint RT-PCR and ELISA assays. Use of updated assays will improve biosecurity protocols as well as the study of viruses infecting pollen.

Pollen transmission of asparagus virus 2 (AV-2) may facilitate mixed infection by two AV-2 isolates in asparagus plants.

Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus and thought to induce the asparagus decline syndrome. AV-2 is known to be transmitted by seed, and the possibility of pollen transmission was proposed 25 years ago but not verified. In AV-2 sequence analyses, we have unexpectedly found mixed infection by two distinct AV-2 isolates in two asparagus plants. Because mixed infections by two related viruses are normally prevented by cross protection, we suspected that pollen transmission of AV-2 is involved in mixed infection. Immunohistochemical analyses and in situ hybridization using AV-2-infected tobacco plants revealed that AV-2 was localized in the meristem and associated with pollen grains. To experimentally produce a mixed infection via pollen transmission, two Nicotiana benthamiana plants that were infected with each of two AV-2 isolates were crossed. Derived cleaved-amplified polymorphic sequence analysis identified each AV-2 isolate in the progeny seedlings, suggesting that pollen transmission could indeed result in a mixed infection, at least in N. benthamiana.

Development of molecular tests for the detection of ILAR and latent viruses in fruit trees.

The detection throughout the year of latent and ILAR viruses in fruit tress by classical serological tests appear to be unreliable. We have developed RT-PCR tests for a reliable detection of latent and ILAR viruses in fruit trees. These assays were then simplified to allow the direct use of crude plant extracts instead of total RNA preparations, and the analyses of pooled samples. In this way, such RT-PCR protocols are suitable for a routine diagnosis of latent and ILAR viruses in fruit tree certification.