Philippe JEAN-PIERRE

Background Faced with the recrudescence of viral CRESS-DNA plant diseases, the availability of efficient and costeffective tools for routine diagnosis and genomic characterisation is vital. As these viruses possess circular singlestrand DNA genomes, they have been routinely characterised using rolling circle amplification (RCA) coupled with Sanger sequencing. However, while providing the basis of our knowledge of the diverse CRESS-DNA viruses, this approach is laboratory-intensive, time-consuming and ultimately ineffective faced with co-infection or viruses with multiple genomic components, two common characteristics of these viruses. Whereas alternatives have proved effective in some applications, there is a strong need for next-generation sequencing methods suitable for small-scale projects that can routinely produce high quality sequences comparable to the gold standard Sanger sequencing.

Here, we present an RCA sequencing diagnostic technique using the latest Oxford Nanopore Technology flongle flow cells. Originally, using the tandem-repeat nature of RCA products, we were able to improve the quality of each viral read and assemble high-quality genomic components. The effectiveness of the method was demonstrated on two plant samples, one infected with the bipartite begomovirus African cassava mosaic virus (ACMV) and the other infected with the nanovirus faba bean necrotic stunt virus (FBNSV), a virus with eight genomic segments. This method allow us to recover all genomic components of both viruses. The assembled genomes of ACMV and FBNSV shared 100% nucleotide identity with those obtained with Sanger sequencing. Additionally, our experiments demonstrated that for similar-sized components, the number of reads was proportional to the segment frequencies measured using qPCR.

In this study, we demonstrated an accessible and effective Nanopore-based method for high-quality genomic characterisation of CRESS-DNA viruses, comparable to Sanger sequencing. Face with of increasing challenges posed by viral CRESS-DNA plant diseases, integrating this approach into routine workflows could pave the way for more proactive responses to viral epidemics.

In tropical humid Africa, sandy soils under periodically burnt herbaceous savannas exhibit generally low carbon (C) content and nitrogen (N) availability. Savanna afforestation may overcome these limitations through changes in soil functioning, yet these processes still need to be explored. In this study, we investigated whether changes in the composition of soil micro-food web may explain soil C and N cycling increases following savanna afforestation. We conducted a 7-year experiment in Congo including Eucalyptus and N 2 -fixing Acacia monocultures and Eucalyptus-Acacia mixtures established on former herbaceous savannas. We assessed in each of these modalities the soil attributes: organic C and N, pH H2O , nitrate, ammonium, net C and N mineralization and nitrification rates, along with the abundances of bacteria, fungi, nematodes, ammonia-oxidizing archaea (AOA) and bacteria (AOB) in the top 10 cm layer. Afforestation of savannas with Eucalyptus for timber production increased soil C by 1.7 times, soil net N mineralization rates by 1.9 times and soil inorganic nitrogen by 2.5 times. Mixed Acacia-Eucalyptus and Acacia monoculture plantations further improved the rate of net nitrogen mineralization by a factor of 1.4 and soil inorganic N by a factor of 2.3 compared with Eucalyptus monocultures. These changes were associated with a gradual increase in AOA abundance from savanna to Eucalyptus monoculture, Eucalyptus-Acacia mixtures and finally Acacia monoculture. Savanna afforestation resulted in a significant increase in the absolute abundance of bacterial-feeding nematodes by 678 %, but to a decrease in the abundance of both fungal-feeders, and omnivores and predators. Increase in N cycling was positively associated with both AOA and nematode bacterial-feeder abundances, underlining the importance of monitoring micro-food web structure to understand better how land use changes affect soil biogeochemical cycling in the context of tropical afforestation.