Complete Genome Sequence of a Novel Umbra-like Mycovirus From the Plant Pathogenic Fungus Phoma Matteucciicola

Here, a novel umbra-like mycovirus, ‘Phoma matteucciicola RNA virus 2’ (PmRV2), isolated from Phoma matteucciicola strain HNQH1 in Hainan province of China, was sequenced and analyzed. The complete genomic sequence of PmRV2 is 3,460 nucleotides (nts) with a GC content of 56.71%. Sequence analysis of PmRV2 indicated that the presence of two noncontiguous open reading frames (ORFs) encoding a hypothetical protein and a RNA-dependent RNA polymerase (RdRp), respectively. PmRV2 contains a metal-binding ‘GDN’ triad in Motif C of RdRp while most + ssRNA mycoviruses contained a ‘GDD’ motif in the same region. Additionally, a BLASTp search showed that the RdRp amino acid sequence of PmRV2 was most closely related to the RdRp of Macrophomina phaseolina umbra-like virus 1 (50.72% identity) and Erysiphe necator umbra-like virus 2 (44.84% identity). Phylogenetic analysis indicated that PmRV2 grouped together with Erysiphe necator umbra-like virus 2 (EnUlV2) within the recently proposed family of ‘Mycotombusviridae’.


Introduction
Mycovirus (fungal viruses) are viruses that can infect and replicate in phytopathogenic fungi, yeasts, or oomycetes [1,2]. In the past few decades, the number of recognized fungal viruses has rapidly increased with the development and wide usage of next-generation sequencing technologies [3,4]. Recently, over 300 mycoviral sequences have been recorded in the National Center for Biotechnology Information (NCBI) database, which are divided into 19 families and one unclassi ed genus by NCBI [5,6]. Mycoviruses are mainly classi ed according to the viral genome segments and replication mode. The genomes of mycoviruses are composed of double-stranded RNA (dsRNA), positive-sense single-stranded RNA (+ssRNA), negative-sense single-stranded RNA (-ssRNA), or single-stranded DNA (ssDNA) [7-9].
Mycoviruses with a +ssRNA genome are classi ed into nine families, including Alpha exiviridae, Barnaviridae, Botourmiaviridae, Delta exiviridae, Gamma exiviridae, Hypoviridae, Endornaviridae, Narnaviridae, and the proposed family 'Mycotombusviridae' [10]. Although the vast majority of mycoviruses cause cryptic infections, the infection of some mycoviruses causes obvious phenotypic alterations in growth, sporulation, pigmentation, and virulence, which often result in hypovirulence and debilitation [11]. Mycovirus-mediated hypovirulence generally has the potential for biological control of plant pathogenic fungus diseases. For instance, Cryphonectria hypovirus 1 (CHV1) was successfully used as a biological control agent to control chest blight disease in Europe in the last century [12].
Phoma matteucciicola is a destructive pathogenic fungus of Curcuma wenyujin causing leaf blight disease in Hainan, China [6, 10,11]. With respect to C. wenyujin-infecting P. matteucciicola, only three viruses, Phoma matteucciicola ourmia-like virus 1 (PmOLV1) [6], Phoma matteucciicola RNA virus 1 (PmRV1) [10], and Phoma matteucciicola partitivirus 1 (PmPV1) [13], have been reported. In the present study, we describe a novel umbra-like mycovirus being isolated from P. matteucciicola strain HNQH1. The virus is tentatively named Phoma matteucciicola RNA virus 2 (PmRV2), and proposed to be a new member within the recently proposed family of 'Mycotombusviridae'. Provenance of the virus material P. matteucciicola strain HNQH1 was originally isolated from C. wenyujin, showing symptoms of leaf blight disease in Hainan, China, in 2018, and identi ed as P. matteucciicola based on morphological characteristics and molecular phylogeny [14]. The mycelia of HNQH1 were cultured for 7 days on cellophane membranes placed on top of potato dextrose agar (PDA) plates for dsRNA extraction. The viral dsRNA of HNQH1 was extracted from approximately 2.0 g fresh mycelia, using phenol-chloroformethanol method [15]. After extraction, the crude dsRNAs were puri ed with DNase I and S1 nuclease (Takara) digestions, respectively.
The cDNA library was constructed with a RevertAid First Strand cDNA Synthesis Kit (Thermo) using tagged oligonucleotide (5'-CGATCGATCATGATGCAATGC-3') [16]. The reverse transcription-polymerase chain reaction (RT-PCR) products were puri ed with a gel extraction kit (Omega) and cloned into pMD19-T vector (Takara) for Sanger sequencing. Designing speci c primers based on the obtained cDNA sequences, the internal gaps between the clones were obtained. To obtain the termini sequences of PmRV2, rapid ampli cation of cDNA ends (RACE) were performed as previously described [10]. The resulting sequence of PmRV2 was deposited in the GenBank database under the accession no.

MW970051.
The virus genome sequence analysis, including sequence assembling and ORF prediction, was carried out using DNAMAN software package (version 6.0). RNA structures of the terminal sequence were ORF2 is 1497 nt long and encodes a 498 aa protein with a predicted molecular mass of 56.1 kDa.
Database searches showed that ORF2 was most closely related to the RdRps of Macrophomina phaseolina umbra-like virus 1 (50.72% identity) and Erysiphe necator umbra-like virus 2 (44.84% identitiy), respectively. In addition, the PmRV2 RdRp has a GDN triad in motif C, while the GDD motif was usually found in +ssRNA viruses ( Figure 1C). Previous study showed that modi cation of GDD to GDN has an adverse impact on enzymatic activity in +ssRNA viruses [20].
To dissect the relationship between PmRV2 and other mycoviruses, a molecular phylogenetic tree was constructed based on the amino acid sequences of the RdRp regions of PmRV2, twelve reported tombusviruses, and other ve reported hypoviriruses and narnaviriruses. The ML tree revealed that