Measuring and mapping the plant virus richness at the ecosystem scale – PHYTOVIRUS

Several methodological approaches regarding viral metagenomics have been undertaken since the beginning of the project.
- The robustness of the complete viral metagenomic protocol (sample drying and grinding; extraction, amplification, sequencing and bioinformatics analysis of sequences) is currently being tested.
- The «Nanopore« sequencing technique has been successfully developed to obtain the complete genomes of single-stranded circular DNA viruses. A scientific paper has been published based on these new protocols. This approach is now used by three partners of the project.
- Successful development of Nanopore sequencing of amplification products from semi-purification of viral nucleic acids (VANA technique)


- The botanical metabarcoding approach based on three markers (ITS, trnL-g and trnL-c) has proven to be effective in identifying plant communities harvested in France.

These methodological advances are encouraging and should enable us to process all the samples collected in order to meet the general objective of WP1.

WP1: Measuring and mapping the phytoviral species richness of ecosystems: testing whether plant species richness influences phytovirus species richness

- Plant collections
Several plant collection campaigns have been carried out in 2020-2021 despite the occurrence of the COVID19 pandemic, here is the detailed list task by task :
- Task1.1: Phytovirus species richness of uncultivated plants within natural ecosystems
- Bordeaux (5 grids), Camargue (5 grids), Réunion (5 grids) in 2020
- Bordeaux (5 grids), Angers (5 grids), Camargue (5 grids) and Réunion (5 grids) in 2021
- Togo (5 grids) in 2021
It should be noted that we could take advantage of the presence of a Togolese Post-Doc (Essowè Palanga) within the UMR PHIM in 2021 to organize and carry out a sampling campaign in Togo. The sampling campaigns planned in Angers (2020), Arizona (2020 and 2021) and South Africa (2020 and 2021) were not carried out because of the pandemic. These collections are therefore postponed to 2022 and 2023.

- Task1.2: Phytovirus species richness at the interfaces between managed cultivated ecosystems and unmanaged natural ecosystems
- 4 grids on plots at the interface cultivated area / natural area in Camargue in 2021
- 4 grids on agricultural plots in the Camargue in 2021

- Task 1.3: Phytovirus species richness within invasive exotic plants
Two sampling protocols have been developed to meet the objective of task 1.3.
- Station-centered« protocol: on a collection site, exotic plants as well as their native congeners are sampled at a rate of 50 plants per botanical species. This protocol was followed in Reunion in 2020 and 2021 and in Camargue in 2021.
Plant-centric« protocol: two invasive alien plants in France were selected (Bothriochloa barbinodis and Senecio inaequidens) as study models. These two plants, as well as native species of the same family, were collected on several sites in France (Bordeaux, Angers, Mediterranean coast) in 2021. Also in 2021, populations of these two plants were also collected in their respective centers of origin (i.e. Arizona for Bothriochloa barbinodis and South Africa for Senecio inaequidens).

WP2: Studying the effect of plant community features on viral species richness
The collection campaigns were carried out as planned in the initial project. Five collections have been completed to date (June 2020, November 2020, March 2021, June 2021 and November 2021), for a total of 480 samples collected. The VANA viral metagenomics protocol has been applied from these samples and an Illumina sequencing run has been performed in 2021 from 288 samples. The sequences are currently being analyzed.
The progress of this WP is in accordance with the initial plan.

The delays observed for all the tasks result from the occurrence of the COVID19 pandemic and the sanitary restrictions that prevailed in 2020 and 2021 (confinements, difficulty in carrying out field missions, border closures etc.). This series of difficulties led us to request a one-year postponement of the end of the project (end of February 2025), a request that was accepted by the ANR.

Task 2.2 («Effects of plant community composition, species richness, plant biomass and population density on the species richness of phytoviruses of an exotic invasive plant«) could not be carried out because the seeds of Bothriochloa barbinodis sown in the various plant communities installed on the Tour du Valat plot in the Camargue did not germinate. A second sowing campaign was carried out in February 2020, and again no seeds germinated. This task was therefore abandoned. However, Bothriochloa barbinodis was chosen as one of two study models (Task 1.3) seeking to compare the virome of invasive alien plants with the viromes of botanically related native plants.

Apart from the (probably unresolved) problem of the COVID19 pandemic, the schedule and budget are under control (thanks in particular to the acceptance of a one-year postponement of the project closure by the ANR). The partners collaborate in accordance with the expectations and the established planning. The relations between partners are very good and very constructive.

Two protocols using the Nanopore sequencing approach to obtain the complete genomes of geminiviruses were developed in the Réunion and Montpellier laboratories. This important result was published in the journal Microorganisms.

Emergent diseases of plants, a high proportion of which are caused by phytoviruses, are a significant burden on the food security and economic stability of societies. However, no studies have provided a comprehensive view of the geographical distribution of phytovirus diversity to date, including both the numbers or richness of virus species and the evenness of their distribution in any individual environment on Earth. Our capacity to detect phytoviruses in the early phases of emergence is strongly dependent on our ability to determine the frequencies and geographical distributions of both new introductions of virus and plant species to environments, and the new virus-host encounters that ensue within these environments. This gap in our knowledge undermines our understanding of virus adaptation and limits our capacity to derive truly general predictive models of phytovirus emergence.

Recent viral metagenomics studies, which have leveraged methodological innovations to achieve the relatively unbiased sampling and sequencing of viral genomes within natural environments have paved the way towards the analyses of phytovirus biodiversity in these environments in sufficient detail to drive major advances in our understanding of the evolutionary processes that underlie the emergence of phytoviruses as agricultural pathogens. These studies have already revealed that uncultivated areas within agricultural settings are key-players in the ecology and evolution of agriculturally relevant phytoviruses. The two overarching hypotheses that we propose testing in this project will extend the findings of these pioneering virus biodiversity studies:

- Plant community structure influences phytoviral community structure: We hypothesize that plant community species richness, composition, density and biomass are predictors of phytovirus species richness.
- The rate of molecular evolution of viruses is slower in uncultivated areas than in cultivated areas: We hypothesize that land uses changes and cropping practices are likely to select for fast-growing, early-transmitted, and more virulent viruses.

The PHYTOVIRUS project has three scientific and technological objectives that will aim at testing whether plant species richness influences phytovirus species richness in natural and cultivated areas (objective 1), studying experimentally the effect of plant communities on phytoviral species richness (objective 2), and searching for evolutionary fingerprints associated with emergence within phytovirus genomes (objective 3). The first work package (WP1) will yield a substantially expanded inventory of known phytoviruses and provide detailed comparative data on the species richness of plants and phytoviruses in several natural unmanaged ecosystems and managed agricultural systems. WP2 will experimentally test whether associations exist between the species richness of phytovirus and plant communities. Finally, WP3 will explore sequence data generated in WP1/2 to detect and characterise evolutionary footprints (evolutionary rates, recombination patterns and natural selection patterns) that are associated with emergence.

Besides providing the first assessments of phytovirus species richness in selected environments, this project also aims at defining and demonstrating a standardized experimental approach to measure phytovirus species richness that could then be universally used at scales ranging from defined ecosystems through to entire continents.

The project brings together research groups that are specialized in plant virology, viral metagenomics, plant ecology, and the computational analysis of virus evolution. This multidisciplinary consortium has the ability to implement a holistic research program that is without equivalent at the international levels with respect to its focus on phytovirus species richness and the plant community and viral evolutionary parameters that have shaped this species richness.