Characterization of a novel mycotombus-like virus from the plant-pathogenic fungus Phoma matteucciicola

Here, we report a novel mycotombus-like mycovirus, tentatively named "Phoma matteucciicola RNA virus 2" (PmRV2), derived from the phytopathogenic fungus Phoma matteucciicola strain HNQH1. The complete PmRV2 genome is comprised of a positive-sense single-stranded RNA (+ssRNA) of 3,460 nucleotides (nt) with a GC content of 56.71%. Sequence analysis of PmRV2 indicated the presence of two noncontiguous open reading frames (ORFs) encoding a hypothetical protein and an RNA-dependent RNA polymerase (RdRp), respectively. PmRV2 contains a metal-binding ‘GDN’ triplet in motif C of RdRp, while most +ssRNA mycoviruses contained a ‘GDD’ motif in the same region. 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 (EnUlV2, 44.84% identity). Phylogenetic analysis indicated that PmRV2 grouped together with EnUlV2 within the recently proposed family "Mycotombusviridae".

Mycoviruses are fungal viruses that can infect and replicate in phytopathogenic fungi, yeasts, or oomycetes [1,2]. In the past few decades, the recognition of novel fungal viruses has increased rapidly with the development and wide usage of next-generation sequencing technologies [3,4]. Over 300 mycoviral sequences have been recorded in the National Center for Biotechnology Information (NCBI) database, and these are divided into 22 families (https:// talk. ictvo nline. org/) [5,6]. Mycoviruses are mainly classified according to their viral genome structure 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, rarely, single-stranded DNA (ssDNA) [7][8][9]. Mycoviruses with a +ssRNA genome are classified into nine established families (Alphaflexiviridae, Barnaviridae, Botourmiaviridae, Deltaflexiviridae, Gammaflexiviridae, Hypoviridae, Endornaviridae, Narnaviridae, and Mitoviridae) as well as the proposed family "Mycotombusviridae" [10]. Although the vast majority of mycoviruses cause cryptic infections, infection by some mycoviruses causes obvious phenotypic alterations in fungal growth, sporulation, pigmentation, and virulence, which often result in hypovirulence and debilitation [11]. Mycovirus-mediated hypovirulence generally has the potential to be exploited for biological control of fungal diseases of plants. For instance, Cryphonectria hypovirus 1 (CHV1) was used successfully in the last century as a biological agent to control chestnut blight disease in Europe [12,13].

Provenance of the virus material
P. matteucciicola strain HNQH1 was originally isolated in 2018 from C. wenyujin showing symptoms of leaf blight disease in Hainan, China, and identified as P. matteucciicola based on morphological characteristics and phylogenetic analysis [15]. 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 was extracted from approximately 2.0 g of fresh mycelia of strain HNQH1, using selective absorption to columns of CF-11 cellulose powder in the presence of 16% ethanol [16]. After extraction, the crude dsRNA was purified using DNase I and S1 nuclease (Takara) digestion (Fig. 1A).
A cDNA library was constructed using a RevertAid First Strand cDNA Synthesis Kit (Thermo), using the tagged oligonucleotide 5′-CGA TCG ATC ATG ATG CAA TGC-3′ [17]. The reverse transcription polymerase chain reaction (RT-PCR) products were purified using a gel extraction kit (Omega) and cloned into the vector pMD19-T (Takara) for Sanger sequencing. Specific primers were designed based on the cDNA sequences obtained to fill in the internal gaps between the cloned sequences. To obtain the terminal sequences of PmRV2, rapid amplification of cDNA ends (RACE) was performed as described previously [10]. The resulting sequence of PmRV2 was deposited in the GenBank database under the accession no. MW970051.
ORF1 is 870 nt long and encodes a hypothetical protein of 289 aa with a predicted molecular mass of 30.1 kDa (Fig. 1B). A BLASTp search showed that the aa sequence of the protein encoded by PmRV2 ORF1 shares 50.29%, 40.74%, and 42.63% sequence identity with those of Erysiphe necator umbra-like virus 2, Macrophomina phaseolina umbra-like virus 1, and Macrophomina phaseolina umbralike virus 3, respectively.
ORF2 is 1497 nt long and encodes a protein of 498 aa 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% identity). The PmRV2 RdRp has a GDN triplet in motif C, whereas a GDD motif is more commonly found in +ssRNA mycoviruses (Fig. 1D). A previous study showed that modification of GDD to GDN has an adverse effect on the enzymatic activity of RNA polymerases of +ssRNA viruses [21].
To assess the phylogenetic relationship of PmRV2 to other mycoviruses, a phylogenetic tree was constructed based on the amino acid sequences of the RdRps of PmRV2, 12 tombusviruses, and five other hypoviruses and narnaviruses. The ML tree revealed that PmRV2 grouped together with Erysiphe necator umbra-like virus 2 within a recently proposed family of mycotombus-like viruses (Fig. 2). PmRV2 was also in the same cluster as Phoma matteucciicola RNA virus 1 (PmRV1), a mycovirus of P. matteucciicola that was identified previously in our laboratory [10], indicating that these two viruses have a close phylogenetic relationship. Therefore, PmRV2 should be considered a new member of the recently proposed family 'Mycotombusviridae'.