Abstract The obligatory biotrophic oomycetes Plasmopara viticola is the causal agent of downy mildew, a destructive disease of grapevine worldwide. So far, chemical fungicides are widely employed to limit this pathogen, but their adverse effects are stimulating the quest for environmentally friendly alternative approaches. Here, we report on the search for mycoviruses that might be later developed as biocontrol agents for this pathogen. Symptomatic leaves were collected from various regions in Spain and Italy and mycelia associated to leaf lesions was harvested. Total RNA extractions were depleted of rRNA and metatranscriptomes were generated using a high-throughput sequencing approach. The virome associated to leaf lesions was then characterized through a bioinformatic pipeline relying on blast searches against current viral databases. Here, we present an inventory of 283 new RNA viruses: 222 positive strand RNA viruses, 29 negative strand RNA viruses, 27 double-stranded RNA viruses and 5 ORFan virus RdRP segments, which could not be reliably assigned to any existing group in the Riboviria. In addition to ORFan viruses, we found other surprising new evolutionary trajectories in this wide inventory of viruses. The most represented viruses in our collection are those in phylum Lenarviricota, and, among them, a group of mycovirus segments distantly related to narnaviruses, but characterized by a polymerase palm domain lacking subdomain C, with the putative GDD catalytic triad. We also provided evidence of a strict association between two RNA segments that form a new mycovirus clade of positive strand RNA in the phylum Kitrinoviricota, order Martellivirales. In the phylum Negarnaviricota, we report for the first time in the order Mononegavirales a clade of viruses that is ambisense, a feature that so far was present only in the order Bunyavirales. Furthermore, in the same phylum we detected the widespread occurrence and abundant accumulation in our libraries of a distinct mycovirus clade distantly related to the Muvirales and Goujanvirales orders, which so far include only viruses infecting invertebrates. Possible new oomycetes-specific virus clades are also described in the phylum Duplornaviricota. These data greatly expand the evolutionary history of mycoviruses adding new layers of diversity to the realm Riboviria.

中文翻译：

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与葡萄霜霉病病变相关的病毒组分析揭示了新的真菌病毒谱系

摘要 必需的生物营养卵菌Plasmopara viticola 是全球葡萄藤病害霜霉病的致病菌。到目前为止，化学杀真菌剂被广泛用于限制这种病原体，但它们的不利影响正在刺激对环境友好的替代方法的探索。在这里，我们报告了寻找可能后来开发为这种病原体的生物防治剂的真菌病毒。从西班牙和意大利的不同地区收集有症状的叶子，并收获与叶子病变相关的菌丝体。总 RNA 提取物去除了 rRNA，并使用高通量测序方法生成了元转录组。然后通过依赖于针对当前病毒数据库的爆炸搜索的生物信息学管道来表征与叶片病变相关的病毒组。这里，我们提供了 283 种新 RNA 病毒的清单：222 种正链 RNA 病毒、29 种负链 RNA 病毒、27 种双链 RNA 病毒和 5 个 ORFan 病毒 RdRP 片段，这些片段无法可靠地分配给 Riboviria 中的任何现有组。除了 ORFan 病毒之外，我们还在这个广泛的病毒库中发现了其他令人惊讶的新进化轨迹。我们收集的最具代表性的病毒是 Lenarviricota 门中的病毒，其中一组真菌病毒片段与 narnaviruses 关系较远，但其特征是聚合酶手掌结构域缺乏亚结构域 C，具有推定的 GDD 催化三联体。我们还提供了两个 RNA 片段之间严格关联的证据，这些片段在 Kitrinoviricota 门（Martellivirales）中形成了一个新的正链 RNA 真菌病毒进化枝。在 Negarnaviricota 门中，我们第一次在单倍体病毒目中报告了一个具有歧义的病毒进化枝，这一特征迄今为止仅存在于布尼亚病毒目中。此外，在同一个门中，我们检测到与 Muvirales 和 Goujanvirales 目远相关的独特真菌病毒进化枝的文库中广泛存在和大量积累，迄今为止仅包括感染无脊椎动物的病毒。Duplornaviricota 门也描述了可能的新的卵菌特异性病毒进化枝。这些数据极大地扩展了真菌病毒的进化历史，为 Riboviria 领域增加了新的多样性层次。在同一门中，我们检测到与 Muvirales 和 Goujanvirales 目远相关的独特真菌病毒进化枝在我们的文库中广泛存在和大量积累，迄今为止仅包括感染无脊椎动物的病毒。Duplornaviricota 门也描述了可能的新的卵菌特异性病毒进化枝。这些数据极大地扩展了真菌病毒的进化历史，为 Riboviria 领域增加了新的多样性层次。在同一门中，我们检测到与 Muvirales 和 Goujanvirales 目远相关的独特真菌病毒进化枝在我们的文库中广泛存在和大量积累，迄今为止仅包括感染无脊椎动物的病毒。Duplornaviricota 门也描述了可能的新的卵菌特异性病毒进化枝。这些数据极大地扩展了真菌病毒的进化历史，为 Riboviria 领域增加了新的多样性层次。