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

doi:10.21203/rs.3.rs-2331635/v1

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

https://doi.org/10.21203/rs.3.rs-2331635/v1

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published 09 Mar, 2023

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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 3,460 nucleotides (nts), a positive-sense single-stranded RNA (+ ssRNA) 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 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 (EnUlV2, 44.84% identity). Phylogenetic analysis indicated that PmRV2 grouped together with EnUlV2 within the recently proposed family of ‘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 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 22 families (https://talk.ictvonline.org/) [5, 6]. Mycoviruses are mainly classified 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 rarely single-stranded DNA (ssDNA) [7–9]. Mycoviruses with a + ssRNA genome are classified into nine families, including Alphaflexiviridae, Barnaviridae, Botourmiaviridae, Deltaflexiviridae, Gammaflexiviridae, Hypoviridae, Endornaviridae, Narnaviridae, and the proposed family ‘Mycotombusviridae’ [10]. Although the vast majority of mycoviruses cause cryptic infections, the 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 for biological control of plant pathogenic fungus diseases. For instance, Cryphonectria hypovirus 1 (CHV1) was successfully used as a biological 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, 13]. So far, 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 found in the authors’ laboratory infecting P. matteucciicola and have proved to belong to different virial family, indicating a rich diversity of mycovirus in P. matteucciicola. In this study, we describe a novel mycotombus-like mycovirus 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’ [10].

P. matteucciicola strain HNQH1 was originally isolated from C. wenyujin, showing symptoms of leaf blight disease in Hainan, China, in 2018, and identified as P. matteucciicola based on morphological characteristics and phylogeny analysis [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 was extracted from approximately 2.0 g fresh mycelia of strain HNQH1, using the selective absorption to columns of cellulose powder CF-11 in the presence of 16% ethanol method [15]. After extraction, the crude dsRNAs were purified with DNase I and S1 nuclease (Takara) digestions, respectively.

A 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 purified with a gel extraction kit (Omega) and cloned into pMD19-T vector (Takara) for Sanger sequencing. Designing specific primers based on the obtained cDNA sequences, the internal gaps between the clones were obtained. To obtain the terminal sequences of PmRV2, rapid amplification 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 folding structures of the terminal sequences were predicted using the Mfold (http://mfold.rna.albany.edu/?q=mfold/RNAFolding-Form2.3)[17]. Multiple sequence alignments of the RdRp sequences were performed with Clustal-X [18]. Phylogenetic tree was constructed using the maximum-likelihood (ML) method in Molecular Evolutionary Genetics Analysis (MEGA)-X program with 1,000 bootstrap replicates [18, 19]. Motif searches were performed in conserved domain database (CDD) (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).

The complete genome sequence of PmRV2 is 3460 nucleotides (nt) with a GC content of 56.71%. The 5’- and 3’- untranslated regions (UTRs) of the sequence were 549 nt and 421 nt, respectively (Fig. 1A). Two potential stem-loop structures could be predicted in 5’- and 3’- UTRs with initial ∆G values of -9.90 kcal/mol and − 15.40 kcal/mol, respectively (Fig. 1B). The PmRV2 genome contains two noncontiguous open reading frames (ORFs), referred to as ORF1 (nt 550–1419) and ORF2 (nt 1543–3039) (Fig. 1A).

ORF1 is 870 nt long and encodes a 289 aa hypothetical protein with a predicted molecular mass of 30.1 kDa (Fig. 1A). A BLASTp search showed that the aa sequence of PmRV2 ORF1 shares 50.29%, 40.74%, 42.63% sequence identities with that of Erysiphe necator umbra-like virus 2, Macrophomina phaseolina umbra-like virus 1, and Macrophomina phaseolina umbra-like virus 3, respectively.

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 mycoviruses (Fig. 1C). Previous study showed that modification of GDD to GDN has an adverse impact on enzymatic activity in + ssRNA viruses [20].

To dissect the phylogenetic 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 five other hypoviruses and narnaviruses. The ML tree revealed that PmRV2 grouped together with Erysiphe necator umbra-like virus 2 within the recently proposed family of mycotombus-like viruses (Fig. 2). In addition, PmRV2 was placed in the same cluster with Phoma matteucciicola RNA virus 1(PmRV1), a mycovirus of P. matteucciicola previously identified in our laboratory [10], indicating that these two viruses have a close phylogenetic relationship. Therefore, PmRV2 should be considered a new member within the recently proposed family ‘Mycotombusviridae’.

Conflict of interest All authors declare no conflict of interest.

Ethical approval This article does not contain any studies with animals or human participants performed by any of the authors.

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Journal Publication

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You are reading this latest preprint version

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

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