Molecular characterization of two new alternaviruses identified in members of the fungal family Nectriaceae.

Since the first report in 2009, at least ten additional viruses have been identified and assigned to the proposed virus family Alternaviridae. Here we report two new mycoviruses tentatively assigned to this family, both identified as members of the fungal family Nectriaceae, which were isolated from surface-disinfected apple roots (Malus x domestica, Borkh.) affected by apple replant disease (ARD). ARD is a highly complex, worldwide-occurring disease resulting from plant reactions to a disturbed (micro)-biome and leads to high economic losses every year. The first alternavirus characterized in this study was identified in a Dactylonectria torresensis isolate. The virus was tentatively named dactylonectria torresensis alternavirus 1 (DtAV1) as the first member of the proposed new species Alternavirus dactylonectriae. The second virus was identified in an isolate of Ilyonectria robusta and was tentatively named ilyonectria robusta alternavirus 1 (IrAV1) as the first member of the proposed new species Alternavirus ilyonectriae. Full genomic sequences of the viruses were determined and are presented. Further, we found hints for putative components of a methyl transferase machinery using in silico approaches. This putative protein domain is encoded by segment 2. However, this result only establishes the basis for subsequent studies in which the function must be confirmed experimentally in vitro. Thus, this is the first study where a function is predicted to all three genomic segments within the group of the alternaviruses. These findings provide further insights into the virome of ARD-associated fungi and are therefore another brick in the wall of understanding the complexity of the disease.

First report of a chrysovirus infecting a member of the fungal genus Ilyonectria.

The fungus Ilyonectria pseudodestructans belongs to the family Nectriaceae and was found to be part of the endophytic microbiome of apple trees (Malus x domestica, Borkh.) with apple replant disease (ARD). After dsRNA extraction, a mycoviral infection became evident. Here, we report the identification of a new virus, tentatively named "Ilyonectria pseudodestructans chrysovirus 1" (IpCV1), as the first member of the proposed new species "Alphachrysovirus ilyonectriae" within the genus Alphachrysovirus. This is the first report of a chrysovirus infecting a member of the fungal genus Ilyonectria. IpCV1 has a tripartite dsRNA genome with a total length of 8944 bp. The segments are 3439 bp, 2850 bp, and 2655 bp in length, and each dsRNA carries a single ORF. The encoded viral proteins are a 125.92-kDa RNA-dependent RNA polymerase, a 100.75-kDa coat protein, and one protein of unknown function with a predicted molecular mass of 93.04 kDa. The 5´ and 3´ UTRs are comparatively short and are 79 to 91 bp and 62 to 148 bp in length, respectively. This study provides the basis for further investigations of the impact of IpCV1 on its host and the etiology of ARD.

Three new mycoviruses identified in the apple replant disease (ARD)-associated fungus Rugonectria rugulosa.

In this study, three new mycoviruses were identified co-infecting the apple replant disease (ARD)-associated root endophyte Rugonectria rugulosa. After dsRNA extraction, six viral fragments were visualized. Four fragments belong to a quadrivirus, which has a genome size of 17,166 bp. Each of the fragments of this quadrivirus has a single ORF encoding a protein. Two of these proteins are coat protein subunits, one ORF encodes the RdRp, and one protein has an unknown function. This virus was tentatively named rugonectria rugulosa quadrivirus 1 (RrQV1) as a member of the proposed new species Quadrivirus rugonectria. Another fragment represents the dsRNA intermediate form of a + ssRNA mitovirus with a genome size of 2410 nt. This virus encodes an RdRp and is tentatively called rugonectria rugulosa mitovirus 1 (RrMV1). RrMV1 is suggested as a member of a new species with the proposed name Mitovirus rugonectria. The sixth fragment belongs to the genome of an unclassified dsRNA virus tentatively called rugonectria rugulosa dsRNA virus 1 (RrV1). The monopartite dsRNA genome of RrV1 has a length of 8964 bp and contains two ORFs encoding a structure/gag protein and an RdRp. Full genomic sequences were determined and the genome structure as well as molecular properties are presented. After phylogenetic studies and sequence identity analyses, all three isolates are proposed as new mycoviruses. The results help to improve the understanding of the complexity of the factors involved in ARD and support the interest in mycoviral research. Subsequent analyses need to focus on the impact of mycoviruses on the biology and pathogenicity of ARD-associated fungi. The results of such studies could contribute to the development of mitigation strategies against the disease.