Significance: Virus cross-infection is an important topic in understanding the course of virus dissemination and evolution. Viruses may spread between the same host species or into taxonomically distinct organisms. The occurrences of cross-kingdom viral infection for certain virus groups are suggested by the current virus taxonomic data. In particular, several plants and fungal viruses show close phylogenetic relationships, but productive transmission of virus between plant and fungal hosts in nature has not been directly demonstrated. Here, we describe the natural infection of Rhizoctonia solani fungus by a plant virus, cucumber mosaic virus (CMV). We further demonstrate that R. solani can acquire and transmit CMV during plant infection. Our findings are evidence of cross-kingdom virus transmission from the plant to fungus.

Viroids are pathogenic agents that have a small, circular noncoding RNA genome. They have been found only in plant species; therefore, their infectivity and pathogenicity in other organisms remain largely unexplored. In this study, we investigate whether plant viroids can replicate and induce symptoms in filamentous fungi. Seven plant viroids representing viroid groups that replicate in either the nucleus or chloroplast of plant cells were inoculated to three plant pathogenic fungi, Cryphonectria parasitica, Valsa mali, and Fusarium graminearum. By transfection of fungal spheroplasts with viroid RNA transcripts, each of the three, hop stunt viroid (HSVd), iresine 1 viroid, and avocado sunblotch viroid, can stably replicate in at least one of those fungi. The viroids are horizontally transmitted through hyphal anastomosis and vertically through conidia. HSVd infection severely debilitates the growth of V. mali but not that of the other two fungi, while in F. graminearum and C. parasitica, with deletion of dicer-like genes, the primary components of the RNA-silencing pathway, HSVd accumulation increases. We further demonstrate that HSVd can be bidirectionally transferred between F. graminearum and plants during infection. The viroids also efficiently infect fungi and induce disease symptoms when the viroid RNAs are exogenously applied to the fungal mycelia. These findings enhance our understanding of viroid replication, host range, and pathogenicity, and of their potential spread to other organisms in nature. Significance Viroids are the only known autonomously replicating pathogenic agents that do not encode proteins. As viroids are known only to naturally infect plants, their infectivity and pathogenicity in other eukaryotes are largely unexplored. Herein, we demonstrate the stable infection of three viroid species in different plant pathogenic filamentous fungi and show that viroid infection can reduce the growth and virulence of fungi. In addition to successful viroid RNA inoculation of fungal spheroplasts, viroid infection of fungus could occur through viroid transmission from the plant and when viroid RNAs are directly applied to fungal mycelia. These findings are relevant to our understanding of viroid replication, transmission, and pathogenicity.

Viroids are the smallest known infectious agents that are thought to only infect plants. Here, we reveal that several species of plant pathogenic fungi that were isolated from apple trees infected with apple scar skin viroid (ASSVd) carried ASSVd naturally. This finding indicates the spread of viroids to fungi under natural conditions and further suggests the possible existence of mycoviroids in nature. A total of 117 fungal isolates were isolated from ASSVd-infected apple trees, with the majority (85.5%) being an ascomycete Alternaria alternata and the remaining isolates being other plant-pathogenic or -endophytic fungi. Out of the examined samples, viroids were detected in 81 isolates (69.2%) including A. alternata as well as other fungal species. The phenotypic comparison of ASSVd-free specimens developed by single-spore isolation and ASSVd-infected fungal isogenic lines showed that ASSVd affected the growth and pathogenicity of certain fungal species. ASSVd confers hypovirulence on ascomycete Epicoccum nigrum. The mycobiome analysis of apple tree-associated fungi showed that ASSVd infection did not generally affect the diversity and structure of fungal communities but specifically increased the abundance of Alternaria species. Taken together, these data reveal the occurrence of the natural spread of viroids to plants; additionally, as an integral component of the ecosystem, viroids may affect the abundance of certain fungal species in plants. Moreover, this study provides further evidence that viroid infection could induce symptoms in certain filamentous fungi.