Taro (Colocasia esculenta L. Schott) is a perennial root crop in the family Araceae, which is widely planted in tropical and sub-tropical regions. Like other vegetatively propagated plants, viruses easily accumulate in the corms, resulting in serious degradation of varieties. About eight viruses have been found to infect taro, often in mixed infections, of which the most common in China are dasheen mosaic virus (DsMV), cucumber mosaic virus and taro bacilliform CH virus [1-4].
Ningbo city in Zhejiang province is one of the main areas of taro production in China (~1800ha annually). During 2020-2022, a virus survey was conducted among local taro varieties in Ningbo. One sample with virus-like symptoms of mosaic and feather mottle in the leaves was collected at random for identification by high-throughput sequencing (HTS). Total RNA was extracted from the leaves using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and a cDNA library was constructed using a TruSeq RNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions. An Illumina NovaSeq 6000 platform with PE150bp and CLC Genomic Workbench 11 (QIAGEN) were used for sequencing and data analysis with default parameters. A total of 37,767,580 paired-end reads were obtained, and 148,128 contigs (49-31,520 bp) were generated de novo and compared with sequences in the GenBank database using BLASTn or BLASTx. In addition to the commonly-occurring DsMV, 3 contigs mapped to viruses in the family Mitoviridae: 1 contig of 1210nt mapped to cannabis sativa mitovirus 1 (37.3% aa identity; 68% query coverage) and 2 contigs of 719 and 456nt mapped to paris mitovirus 1 (respectively 50.0 and 54.9% aa identity; 99% and 93% query coverage).
The complete genomic sequence of the novel mitovirus was then determined using specific primers to amplify three fragments while the 5′- and 3′-terminal sequences were determined using RACE (Tiosbio, Beijing, China) (Supplementary Table S1). PCR products were then cloned into the pEASY-T5 Zero Cloning Vector (TransGen Biotech, Beijing), and at least 5 clones of each product were sent for Sanger sequencing (Ykang, Hangzhou, China). The complete sequence was assembled using DNAMAN 8.0 software (Lynnon Biosoft, Canada) and indicated the presence of a single mitovirus. The complete 2921nt of this novel virus, named Colocasia esculenta associated mitovirus 1 (CeaMV1) was deposited in the GenBank database under accession OR134096. CeaMV1 has a single ORF (nt 307-2592), which is predicted to encode an 87.2 kDa RNA-dependent RNA polymerase (RdRp) of 761aa, and has 5′ and 3′ untranslated regions (UTRs) of 306bp and 329bp, respectively (Fig. 1a).
A conserved Mitovir_RNA_pol domain (pfam05919) at aa 250-547 of the CeaMV1 RdRp was confirmed by a conserved domain database search. The aa sequence was then aligned with those of all reported plant mitoviruses and some mitoviruses from fungi using MAFFT version 7, employing the L-INS-i algorithm [5]. The five highly conserved motifs (Ι to V) of mitoviruses were all identified in CeaMV1 (Fig. 1b). In a BLASTp search, the CeaMV1 RdRp was most closely related to those of the plant-infecting mitoviruses paris mitovirus 1 (48.0%, 95% query coverage), solanum chacoense mitovirus 1 (44.6%, 98% query coverage) and cannabis sativa mitovirus 1 (45.4%, 95% query coverage). These aa identity values are well below the demarcation criteria currently used to establish new species in the family (<70% aa identity) [6,7].
Phylogenetic analysis based on a multiple sequence alignment of the RdRp domain sequences demonstrated that CeaMV1 is closely related to ParMV1 (Fig. 2), and clusters with other plant-infecting viruses as a distinct clade in the genus Duamitovirus. No fungal infection was detected in the leaf sample in which CeaMV1 was found, further indicating that CeaMV1 is a plant mitovirus. In tests of taro plants from different areas of Ningbo, CeaMV1 was detected in 17 out of the 34 samples suggesting that it is widespread. Further work is needed to determine whether this mitovirus has any direct impact on the growth of taro and to evaluate its economic importance. To our knowledge, this is the first report of the complete genome sequence of a mitovirus associated with C. esculenta.