Viromics Reveals Two Novel Viruses of the Family <i>Closteroviridae</i> in <i>Codiaeum variegatum</i> Plant with Leaf Variegation Symptoms

<i>Codiaeum</i> <i>variegatum</i> is a valuable ornamental plant with distinct bright yellowing and golden spots on dark green leaves, which resemble virus symptoms. To investigate the factors, especially viral agents, associated with the variegated leaf color of <i>C. variegatum</i>, we performed virome profiling of a single <i>C. variegatum</i> 'Gold Dust' leaf sample collected from Hainan, China, using ribosomal RNA-depleted total RNA sequencing on an Illumina NovaSeq 6000 platform. Two novel viruses, with two variants each, belonging to the family <i>Closteroviridae</i>, were detected and characterized: Croton golden spot-associated virus C variants 1 and 2 (CGSaVC-v1 and CGSaVC-v2) of the genus <i>Crinivirus</i> and Croton golden spot-associated virus A variants 1 and 2 (CGSaVA-v1 and CGSaVA-v2) of the genus <i>Ampelovirus</i>. Transmission electron microscopy showed long, flexuous, filamentous virus particles approximately 15 nm in diameter and 760 to 770 nm in length. Molecular screening of 97 variegated individual plant leaves showed a high prevalence of CGSaVA-v2 (90.7%), CGSaVA-v1 (75.3%), CGSaVC-v1 (70.1%), and CGSaVC-v2 (47.4%), while asymptomatic leaves near the meristem tip were mostly free of the target viruses. To our knowledge, this is the first study to demonstrate the significant association between closterovirids and the golden spots. The findings provide novel insights into the genetic diversity of the family <i>Closteroviridae</i> and inform future germplasm conservation and new cultivar development of <i>C. variegatum</i>.