Asynchronous Viral Spread of Two Unrelated Viruses Determines Lettuce Big Vein Disease Symptom Development

Lettuce big-vein disease (LBVD) is a major disease affecting lettuce cultivation worldwide. LBVD is caused by two unrelated negative-stranded RNA viruses, Mirafiori lettuce big-vein virus (MiLBVV; Ophiovirus mirafioriense; Aspiviridae) and lettuce big-vein associated virus (LBVaV; Varicosavirus lactucae; Rhabdoviridae), both vectored by the soilborne fungus Olpidium virulentus. Despite extensive research, a synergistic effect between the two viruses has not been observed, whereas both viruses individually have been suggested to be the causal agent for the disease. By performing lettuce reinfections using a large soil sample collection carrying LBVD-infested O. virulentus spores, the presence of LBVaV was consistently established in diseased lettuce heads, whereas MiLBVV infections were apparently less prevalent. However, aboveground infections with MiLBVV corresponded with strong disease symptoms. Strikingly, the spread of LBVaV from the root to shoot always preceded that of MiLBVV. The LBVaV systemic spread was highly synchronized between plants, whereas MiLBVV spread was always delayed and asynchronous. A pangenome analysis revealed independent segment reassortments for both viruses, indicative of mixed field infections over the sampled period. However, RNA segment abundance was highly conserved for both viruses between all reinfections, suggesting that segment abundance has a regulatory role for the two individual viruses but is not impacted by the presence of the other two viruses. The pangenome analysis also revealed different evolutionary rates of the viral open reading frames, suggesting that mutagenesis of certain open reading frames compromises viral fitness and thus revealing a potential weak spot for both viruses.