EVolution of CO-infecting PAthogens in Rice – EVCOPAR

Three major approaches were used:

- Intensive field surveys with symptom observation and sampling in Burkina Faso

We both benefited from previous surveys, performed in 2015-2019, and performed additional field work in two sites and two consecutive years. A total of 1666 rice samples were collected and analysed (see doi.org/10.1101/2025.03.11.638810)

- Molecular work

We analysed collected samples with a newly designed molecular method detecting simultaneously the virus and the bacteria (see doi.org/10.1101/2025.03.11.638810)

We characterised the genetic diversity of the virus, by partial Sanger sequencing, and nanopore sequencing for the samples that were suspected to be simultaneously infected by two viral isolates (methodology described in the article 10.1371/journal.pone.0311555)

We characterised the genetic diversity of the bacteria though MLVA analysis

- Experimental infections and co-infections

We performed experimental inoculations from collected viral and bacterial isolates. This was done with mono-infections (only one pathogen), mixed infections (two genotypes of the same pathogen) or co-infections (virus-bacteria)

We first benefited from a previous survey (2015-2019) and finalized to monitor the dynamics of the rice yellow mottle virus (RYMV) at local scale. This revealed a high viral diversity, with the current coexistence of various distinct genetic groups at the site scale (ca. 520 ha) and also within various specific fields (25 m side). One genetic lineage, named S1bzn, with a recombinant genome, is the most recently emerged group and increased in frequency over the studied period. Further, we found no significant effect of RYMV genetic groups on symptom expression and viral load, but that infection outcome depended on the specific RYMV isolate, with two isolates from the lineage S1bzn accumulating at the highest level at early infections. Overall, this exemplifies a case of high viral prevalence, high viral diversity, and co-occurrence of divergent genetic lineages at a small geographic scale (see 10.1093/ve/vead049).

Then, in order to document co-occurrences and co-infections for a viral and a bacterial disease of rice in Burkina Faso, we performed a field survey in two sites and two consecutive years (2021 and 2022), combining symptom observations with specific molecular detection. Although only 37 of these samples were annotated in the field as presenting both yellow mottle and BLS symptoms, we show that 166 samples, i.e. 12.4% of all collected symptomatic samples, were co-infected by RYMV and Xanthomonas oryzae. We conclude that symptom observation in the field greatly underestimates co-infection levels. Combining these data with a previously published dataset, we estimated that up to 1-4% of all rice plants could be simultaneously infected by the two pathogens (see 10.1101/2025.03.11.638810 and 10.23708/QB8BOO).

Further, the molecular characterization of the RYMV-positive samples was done, in a first step with partial Sanger sequencing. We found substantial frequency of rice plants infected simultaneously by different RYMV isolates, a pattern confirmed though a protocol of nanopore sequencing and bioinformatic analysis that we set up (see 10.1371/journal.pone.0311555). This method has the benefit to obtain full-length sequences that revealed that evidences of past recombination events in the genomes of various RYMV lineages. Evolutionary consequences of RYMV-RYMV co-infections are suspected, not only because of these past recombination events, but also with the contrasted ranking of some viral isolates when experimentally evaluated in mono-infections vs RYMV-RYMV co-infections.

Finally, we show that the structuration of viral genetic diversity is affected by the site and year, but also that some RYMV lineages are associated with the presence of the bacteria Xo. The latter suggests an influence of the bacteria on the genetic structuration of viral populations, that has to be discussed in light with experimental RYMV-Xo co-infections.

The project, innovative in terms of hypothesis and methodology, yielded significant new results evidencing the impact of multiple infections on pathogen evolution.

Involvement in mentoring and training was high for this project, with a postdoc hired for 2 years, and a total of 8 students (Bsc and Masters) performing internships at IRD-PHIM thanks to the project.

It benefited from in-depth previous and ongoing research on rice pathogens, and also has applications in terms of a better understanding of the dynamics of threats to rice cultivation in West Africa.

We argue that further research is required to document co-circulations of multiple diseases in agro-ecosystems for a better understanding of epidemiology and evolution of pathogen populations.

Two directions are considered for future research:

- Following up epidemiological and evolutionary impact of multiple infections by focusing on multiple viral diseases. Indeed, within another ANR project, we identified new viruses in Burkina Faso, some of them known to affect other crops (10.1094/PBIOMES-08-23-0085-FI; 10.1002/ndr2.70007), and also two completely new viruses (unpublished). Further research is being initiated to develop diagnostic tools and monitor this viral cortege over time to infer their reciprocal potential interactions (both experimentally in greenhouses, and in the field). This research would lead to a better understanding of the factors driving viral dynamics.

- Setting up new tools for pathobiome characterization, with sequencing approach to unravel the global diversity of micro-organisms associated with plants. A project was set up in this respect on bacterial diseases, another on fungal diseases and a third aiming at developing a nanopore sequencing strategy for global assessment (virus, bacteria and fungi) of rice health.

Embracing a ‘multi-pathogen’ approach would be powerful for a more global assessment of plant health.

Pathogens actually share host plants with a myriad of other microbes, some of them being pathogenic. Multiple infections, or ‘co-infection’, occur when a single host plant is infected by various pathogen species, or genotypes. This is known to affect symptom expression and/or pathogen multiplication in various pathosystems. However, the population-scale consequences, in terms of epidemiology and evolution, remain poorly explored in spite of their importance for the control of crop diseases. The EVCOPAR project aims at testing the following hypothesis: Multiple infections impact the evolutionary trajectories of pathogen populations and drive the epidemiological outcome.

EVCOPAR focuses on rice in Sub-Saharan Africa, a crop of increasing importance because of human population growth and change in food habits, and two major rice pathogens : the Rice yellow mottle virus (RYMV) and the bacteria Xanthomonas oryzae (Xo). Our preliminary results showed within-plant interactions between these two pathogens in case of co-infection: the presence of the RYMV virus leads to stronger bacterial symptoms, while the presence of the Xo bacteria reduces viral load. Spatiotemporal surveys of these two diseases have been performed in Burkina Faso, so that samples and detailed information on disease dynamics are already available. EVCOPAR project follows up with the study of population-scale consequences of inter-species (RYMV-Xo) and intra-species (various Xo or RYMV genotypes) co-infections.

The project, subdivided into 3 tasks, proposes an original approach combining field work, experimental infections, viral and bacterial genomics and epidemiological modelling.
In the first task, we will describe the genetic structures of RYMV and Xo populations in disease hotspots through innovative sequencing techniques. The hypothesis is that co-infections affect the population genetic structure of the pathogens involved.
The second task consists in an experimental evolution approach. Viral RYMV populations evolved in mono-infection will be compared with viral populations evolved in co-infection (presence of the Xo bacteria, or of another RYMV genotype), in terms of genomics and phenotype (virulence in susceptible rice and capacity to overcome rice resistance).
Finally, the third task will integrate the data generated in tasks 1 and 2, and develop an epidemiological modelling approach of the pathosystem, specifically addressing the effect of co-infection on epidemiology and evolution of the two considered pathogens.

Overall, the EVCOPAR project takes a ‘pathobiome’ view that integrates each pathogen in the microbial community of its host. Within this framework, the originality of this project resides in a population-scale approach, with particular focus on evolution and epidemiology, and the application to a pathosystem of strong agronomic importance. Consequently, expected results will be disseminated as academic outputs, but will also shed new light for integrated control of crop diseases, and therefore for plant health, a prerequisite for food safety in Sub-Saharan Africa.