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, that is, 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, while both viruses individually have been suggested to be the causal agent for the disease. By performing lettuce infections using a large soil sample collection carrying LBVD infested O. virulentus spores, the presence of LBVaV was consistently established in diseased lettuce heads, while MiLBVV infections were apparently less prevalent. Yet, 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 the plants, while MiLBVV spread was always delayed and less synchronized. A pan-genome analysis revealed independent segment reassortments for both viruses indicative of mixed field infections over the sampled period. Yet, RNA segment abundance was highly conserved for both viruses between all re-infections, suggesting that segment abundance has a regulatory role for the two individual viruses, but that segment abundance is not impacted by the presence of the other two viruses. The pan-genome analysis also revealed different evolutionary rates of the viral ORFs suggesting that mutagenesis of certain ORFs compromises viral fitness and thus revealing a potential weak spot for both viruses.