Diversity of RNA Viruses in Declining Mediterranean Forests

Global change alters forestry habitats and facilitates the entry of new pathogens that do not share a co-evolution history with the forest, leading them into a spiral of decline. As a result, relationships between forest organisms become imbalanced. RNA viruses are of particular concern given their capacity to infect hosts across different kingdoms of life, yet the viromes of Mediterranean forests remain largely unexplored. For this reason, the study of RNA viruses is essential for understanding how viral flow across different hosts might occur, and to prevent possible outbreaks of diseases in the future. In this work, the RNA virus diversity found in trees, arthropods, and fungi from declining Mediterranean forests is described. To this extent, three habitats (Quercus ilex, Castanea sativa and Pinus radiata) were sampled and RNAseq was performed on tree tissues, arthropods, and fungi. In total, 146 viral sequences were detected by searching for matches with conserved motifs of the RNA-dependent RNA polymerase (RdRP) using Palmscan. Up to 15 viral families were identified, with Botourmiaviridae (28.7%) and Partitiviridae (9.6%) being the most abundant. In terms of genome type, ssRNA(+) viruses were the most represented (83.5%), followed by dsRNA (15%) and two ssRNA(-) representatives. Notably, families with documented cross-kingdom capabilities such as Hypoviridae (1), Mitoviridae (6), and Narnaviridae (5) were detected across multiple host types, with one Mitovirus sequence recovered unexpectedly from pine tree tissue. Distribution of viruses across ecosystems included Q. ilex (57.5%), P. radiata (26.7%), and C. sativa (15.8%). Interestingly, two RdRP sequences showed no similarity to any entry in current viral databases, representing potentially novel viruses warranting further characterization. These findings reveal a rich and partially uncharacterized RNA virosphere in declining Mediterranean forests and underscore the importance of multi-host virome surveys for understanding viral flow across kingdoms in threatened ecosystems.