Delayed maturation of nodules reduces symbiotic effectiveness of the Lotus japonicus-Rhizobium sp. NGR234 interaction

Lotus japonicus, a model legume, develops an efficient, nitrogen-fixing symbiosis with Mesorhizobium loti that promotes plant growth. Lotus japonicus also forms functional nodules with Rhizobium sp. NGR234 and R. etli. Yet, in a plant defence-like reaction, nodules induced by R. etli quickly degenerate, thus limiting plant growth. In contrast, nodules containing NGR234 are long-lasting. It was found that NGR234 initiates nodule formation in a similar way to M. loti MAFF303099, but that the nodules which develop on eleven L. japonicus ecotypes are less efficient in fixing nitrogen. Detailed examination of nodulation of L. japonicus cultivar MG-20 revealed that symbiosomes formed four weeks after inoculation by NGR234 are enlarged in comparison with MAFF303099 and contain multiple bacteroids. Nevertheless, nodules formed by NGR234 fix sufficient nitrogen to avoid rejection by the plant. With time, these nodules develop into fully efficient organs containing bacteroids tightly enclosed in symbiosome membranes, just like those formed by M. loti MAFF303099. This work demonstrates the usefulness of using the well-characterized micro-symbiont NGR234 to study symbiotic signal exchange in the later stages of rhizobia-legume symbioses, especially given the large range of bacterial (NGR234) and plant (L. japonicus) mutants that are availabl

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 2013-2020, 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

Identification and Molecular Characterization of a Novel Hordeivirus Associated With Yellow Mosaic Disease of Privet (Ligustrum vulgare) in Europe.

Wild plants serve as a large reservoir of known and yet-unknown viruses and as a source of viral pathogens of cultivated plants. Yellow mosaic disease of forest shrub Ligustrum vulgare (privet) was recurrently observed in Europe for more than 100 years. Using a universal virus identification approach based on deep sequencing and de novo assembly of viral small interfering (si)RNAs we identified a causative agent of this disease in Switzerland and reconstructed its complete 3-segmented RNA genome. Notably, a short 3'-terminal common region (CR) attached to each segment via a ∼53-71 nucleotide poly(A) tract, as determined by RT-PCR sequencing, was initially identified as an orphan siRNA contig with conserved tRNA-like secondary structure. Phylogenomic analysis classified this virus as a novel member in the genus Hordeivirus of family Virgaviridae, which we named ligustrum mosaic virus (LigMV). Similar to other hordeiviruses, LigMV formed rod-shape virions (visualized by electron microscopy), was transmitted through seeds and could also be mechanically transmitted to herbaceous hosts Chenopodium quinoa and Nicotiana benthamiana. Blot hybridization analysis identified genomic and subgenomic RNAs, sharing the 3'-CR and likely serving as monocistronic mRNAs for seven evolutionarily-conserved viral proteins including two subunits of viral RNA-dependent RNA polymerase, coat protein, triple gene block proteins mediating viral movement and cysteine-rich suppressor of RNA silencing. Analysis of size, polarity, and hotspot profiles of viral siRNAs suggested that they are produced by the plant antiviral Dicer-like (DCL) proteins DCL2 and DCL4 processing double-stranded intermediates of genomic RNA replication. Whole genome sequencing of French and Austrian isolates of LigMV revealed its genetic stability over a wide geographic range (>99% nucleotide identity to Swiss isolates and each other), suggesting its persistence and spread in Europe via seed dispersal

Delayed maturation of nodules reduces symbiotic effectiveness of the Lotus japonicus–Rhizobium sp. NGR234 interaction

Lotus japonicus, a model legume, develops an efficient, nitrogen-fixing symbiosis with Mesorhizobium loti that promotes plant growth. Lotus japonicus also forms functional nodules with Rhizobium sp. NGR234 and R. etli. Yet, in a plant defence-like reaction, nodules induced by R. etli quickly degenerate, thus limiting plant growth. In contrast, nodules containing NGR234 are long-lasting. It was found that NGR234 initiates nodule formation in a similar way to M. loti MAFF303099, but that the nodules which develop on eleven L. japonicus ecotypes are less efficient in fixing nitrogen. Detailed examination of nodulation of L. japonicus cultivar MG-20 revealed that symbiosomes formed four weeks after inoculation by NGR234 are enlarged in comparison with MAFF303099 and contain multiple bacteroids. Nevertheless, nodules formed by NGR234 fix sufficient nitrogen to avoid rejection by the plant. With time, these nodules develop into fully efficient organs containing bacteroids tightly enclosed in symbiosome membranes, just like those formed by M. loti MAFF303099. This work demonstrates the usefulness of using the well-characterized micro-symbiont NGR234 to study symbiotic signal exchange in the later stages of rhizobia–legume symbioses, especially given the large range of bacterial (NGR234) and plant (L. japonicus) mutants that are available

High-throughput sequencing for the diagnostic of plant pathologies and identification of pests: recommendations and challenges

International audienceA recommendation – based on reviews by Denise Altenbach and David Roquis – of the article: Massart, S et al. (2022) Guidelines for the reliable use of high throughput sequencing technologies to detect plant pathogens and pests. Zenodo, 6637519, ver. 3 peer-reviewed and recommended by Peer Community in Infections. https://doi.org/10.5281/zenodo.663751

Resistance of Potato Cultivars as a Determinant Factor of Potato virus Y (PVY) Epidemiology

Potato virus Y (PVY) is considered the most economically damaging virus for seed-potato production. PVY isolates are usually divided into three historical PVY strains, namely PVYC, PVYO and PVYN. More recently, recombinant strains named PVYNTN and PVYN-Wi appeared in Europe and North America, rapidly spread into seed-potato production and gradually replaced historical strains. This progression can be explained by several factors, including the differential susceptibility of cultivars to PVY strains. This article presents the results of a Swiss survey of PVY strains conducted in 2012 in which the progressive increase of the prevalence of PVYN-Wi was observed. The occurrence of PVY strains in the two main potato cultivars grown in Switzerland, cv. Agria and cv. Charlotte, is described through the 2012 survey and one additional PVY strain survey conducted in 2014. Both surveys were completed with a mechanical-inoculation assay. The inoculation assay showed that cv. Agria is more susceptible to PVYN-Wi than to PVYNTN, whereas cv. Charlotte is susceptible to both strains. The inoculation assay also showed that the expression of symptoms on cultivars is strain-dependent. These results stress the major role of the resistance profile of cultivars to explain the balance of the PVY strains in potato crops

First detection of ‘Candidatus Phytoplasma ulmi’ in Switzerland and in Orientus ishidae Matsumura, 1902

‘Candidatus Phytoplasma ulmi’ (Ca. P. ulmi) belongs to the ribosomal subgroup 16SrV-A and is associated with dieback, shoot proliferation and yellows disease on various Ulmus spp. Other plant species, such as Carpinus betulus and Prunus spp. have also been reported infected by the same pathogen. In 2021, in the frame of research activities focused on grapevine’s Flavescence dorée (FD), one specimen of Orientus ishidae - an East Palearctic leafhopper that was identified as an alternative vector of FD phytoplasmas - was found harboring Ca. P. ulmi in southern Switzerland. No phytoplasmas were detected in plant samples taken in the same location. Orientus ishidae has already been reported to be able to acquire diverse phytoplasmas associated with other plant diseases, such as Peach X-disease. This is the first report of Ca. P. ulmi in Switzerland, as well as in O. ishidae. Ca. P. ulmi may potentially be present in the wild compartment of the Swiss Pre-alpine and Alpine range, but no dedicated survey has so far been conducted. In the case of O. ishidae, this finding highlights the broad affinity of such a species for the acquisition of several phytoplasmas. This calls for a further investigation regarding its potential role as a vector on various pathosystems of agronomic importance

First detection of ‘Candidatus Phytoplasma ulmi’ in Switzerland and in Orientus ishidae Matsumura, 1902

‘Candidatus Phytoplasma ulmi’ (Ca. P. ulmi) belongs to the ribosomal subgroup 16SrV-A and is associated with dieback, shoot proliferation and yellows disease on various Ulmus spp. Other plant species, such as Carpinus betulus and Prunus spp. have also been reported infected by the same pathogen. In 2021, in the frame of research activities focused on grapevine’s Flavescence dorée (FD), one specimen of Orientus ishidae - an East Palearctic leafhopper that was identified as an alternative vector of FD phytoplasmas - was found harboring Ca. P. ulmi in southern Switzerland. No phytoplasmas were detected in plant samples taken in the same location. Orientus ishidae has already been reported to be able to acquire diverse phytoplasmas associated with other plant diseases, such as Peach X-disease. This is the first report of Ca. P. ulmi in Switzerland, as well as in O. ishidae. Ca. P. ulmi may potentially be present in the wild compartment of the Swiss Pre-alpine and Alpine range, but no dedicated survey has so far been conducted. In the case of O. ishidae, this finding highlights the broad affinity of such a species for the acquisition of several phytoplasmas. This calls for a further investigation regarding its potential role as a vector on various pathosystems of agronomic importance

In situ hybridization of a radioactive RNA probe on resin-embedded legume root-nodule sections: a tool for observing gene expression in the rhizosphere?

In this work we performed in situ hybridization of a carbonic anhydrase RNA $^{35}$S-labeled probe on nodule sections previously embedded in a methacrylate resin. The results were more precise and reproducible than those obtained on paraffin-embedded nodules. Thanks to the small thickness of the sections and the good preservation of tissue during sectioning, the specific localization of the carbonic anhydrase gene expression in the inner cortex of the nodule could be described quite precisely. It is argued that these results are consistent with the hypothesis of osmoregulation of the symbiotic nitrogen fixation. Moreover, the quality of these results with a radioactive probe makes it possible to consider using ISH for such rhizospheric applications as localizing and quantifying a microbial invasion of the endorhizosphere or the rhizoplan by using bacterial- or fungal-specific RNA probes, and counting the number of radioactive dots per cell. However, major artifacts observed with a digoxegin RNA cold probe makes it necessary to recommend the use of a radioactive RNA probe for these prospects.Hybridation in situ d'une sonde ARN radioactive sur des coupes de nodosités de légumineuses incluses dans une résine méthacrylique : un outil pour observer l'expression génique dans la rhizosphère ? Dans ce travail, nous avons réalisé une hybridation in situ d'une sonde ARN d'anhydrase carbonique marquée au $^{35}$S, sur des coupes de nodosités préalablement incluses dans une résine méthacrylique. Grâce à la faible épaisseur des coupes et la bonne conservation des tissus durant la coupe, la localisation spécifique de l'expression du gène d'anhydrase carbonique dans le cortex interne des nodosités a pu être décrite très précisément. Il est argumenté que cette localisation est en accord avec l'hypothèse d'osmorégulation de la fixation symbiotique d'azote. De plus, la qualité de ces résultats avec une sonde radioactive suggère qu'il serait possible d'utiliser l'hybridation in situ pour des applications telles que la localisation et la quantification d'une invasion microbienne de l'endorhizosphère ou du rhizoplan en utilisant des sondes ARN spécifiques de champignons ou de bactéries, et en comptant le nombre de signaux radioactifs par cellule. Il est recommandé d'utiliser un marquage radioactif des sondes, en raison des artefacts observés dans ce travail avec une sonde ARN marquée à la digoxygénine

A novel weevil-transmitted tymovirus found in mixed infection on hollyhock

Abstract Leaves of hollyhock (Alcea rosea) exhibiting vein chlorosis and yellow mosaic symptoms were collected at public sites in Lausanne and Nyon, two cities of western Switzerland. Diagnostic methods untangled in samples from both sites the mixed infections of a novel isometric virus, tentatively named “Alcea yellow mosaic virus” (AYMV) with the carlavirus Gaillardia latent virus. A new potyvirus was also identified in samples from Nyon. A combination of Illumina, Nanopore and Sanger sequencing was necessary to assemble the full-length genome of AYMV, revealing an exceptionally high cytidine content and other features typically associated with members of the genus Tymovirus. The host range of AYMV was found to be restricted to mallows, including ornamentals as well as economically important plants. Phylogenetic analyses further showed that AYMV belongs to a Tymovirus subclade that also gathers the other mallow-infecting members. The virus was readily transmitted by sap inoculation, and the weevil species Aspidapion radiolus was evidenced as a vector. Transmission assays using another weevil or other insect species did not succeed, and seed transmission was not observed