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A novel cytorhabdovirus, tentatively named Actinidia virus D (AcVD), was identified from kiwifruit (Actinidia chinensis) in China using high‐throughput sequencing technology. The genome of AcVD consists of 13,589 nucleotides and is organized into seven open reading frames (ORFs) in its antisense strand, coding for proteins in the order N‐P‐P3‐M‐G‐P6‐L. The ORFs were flanked by a 3′ leader sequence and a 5′ trailer sequence and are separated by conserved intergenic junctions. The genome sequence of AcVD was 44.6%–51.5% identical to those of reported cytorhabdoviruses. The proteins encoded by AcVD shared the highest sequence identities, ranging from 27.3% (P6) to 44.5% (L), with the respective proteins encoded by reported cytorhabdoviruses. Phylogenetic analysis revealed that AcVD clustered together with the cytorhabdovirus Wuhan insect virus 4. The subcellular locations of the viral proteins N, P, P3, M, G, and P6 in epidermal cells of Nicotiana benthamiana leaves were determined. The M protein of AcVD uniquely formed filament structures and was associated with microtubules. Bimolecular fluorescence complementation assays showed that three proteins, N, P, and M, self‐interact, protein N plays a role in the formation of cytoplasm viroplasm, and protein M recruits N, P, P3, and G to microtubules. In addition, numerous paired proteins interact in the nucleus. This study presents the first evidence of a cytorhabdovirus infecting kiwifruit plants and full location and interaction maps to gain insight into viral protein functions. [ABSTRACT FROM AUTHOR]

• Fourteen mycoviruses belonging to 8 families were identified from 83 Alternaria spp. strains that cause the pear black spot disease in Hubei province by High-throughput sequencing (HTS). • Genome sequencing and molecular characterization analysis were conducted on AaCV1, AgMV3, and AgDfV1. • The impact of the mycoviruses on the weakly virulent strain HB-145 was analyzed. Pear black spot (Alternaria spp.) disease widespread in most pear production regions in China, causes early defoliation, which leads to the decline of fruit quality and heavily restricts the healthy development of the pear industry. Presently, no efficient way was found to biocontrol pear black spot disease. The mycovirus-mediated hypovirulent strain is regarded as an important measure of biocontrol of the fungal disease. Therefore, it is particularly urgent to excavate mycoviral resources with hypovirulence in Alternaria spp. strains. This study obtained partial or complete genomes of 14 mycoviruses from 83 Alternaria spp. strains from Wuhan city of China by high throughput sequencing, including 3 double-stranded (ds) RNA and 11 positive single-stranded (+ss) types, which were grouped into 8 distinct lineages, belonging to Chrysoviridae (isolate of AaCV1), Curvulaviridae (one virus), Partitiviridae (one virus), Mitoviridae (three viruses), Botourmiaviridae (five viruses), Deltaflexiviridae (one virus), Fusariviridae (one virus) and Togaviridae (one virus). The multiple mycoviral coinfections were common and prevalent, accounting for 78 % (65/83), with each strain harboring different numbers of mycoviruses, ranging from two to seven in Alternaria spp. Furthermore, the effects of the mycoviruses on the host were analyzed to screen the viruses-inducing hypovirulent strains. The derivative isolates from hypovirulent HB-145 with different mycoviruses were obtained by vertical transmission. It revealed that the mycoviruses have different effects on the virulence of derivative isolates from HB-145. In conclusion, these findings provide important molecular information for the classification, origin, and genetic variety of mycoviruses, and candidate materials for the exploitation of mycoviruses as biocontrol agents. [ABSTRACT FROM AUTHOR]