Molecular characteristics of a novel alternavirus from Trichoderma harzianum

Abstract

We analyzed the dsRNA profiles of Trichoderma spp. and identified a novel mycovirus from Trichoderma harzianum strain NFCF419. Phylogenetic and genomic analysis suggested that this virus is a novel member of the family Alternaviridae. Next-generation sequencing (NGS) analysis revealed that this virus consisted of four genome segments. The complete genome sequences of the segments were determined by NGS, and the NGS results were confirmed by manual sequencing of RT-PCR amplicons using specific primer pairs. 5’- and 3’-RACE was performed to determine the terminal sequences of each segment. The genome segments were found to be 3,572 bp (dsRNA1), 2,552 bp (dsRNA2), 2,593 bp (dsRNA3), and 1,484 bp (dsRNA4) in size. The largest segment (dsRNA1) contains a single open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRP). dsRNA2, 3, and 4 each contain a single ORF encoding a putative methyltransferase, a coat protein, and a hypothetical protein with unknown function, respectively. Evaluation of the genome organization, analysis of the deduced amino acid sequence of the RdRP, and phylogenetic analysis indicated that this virus is a new member of the genus Alternavirus in the family Alternaviridae. Accordingly, we designated this novel mycovirus "Trichoderma harzianum alternavirus 1" (ThAV1). This is the first report of a mycovirus of the family Alternaviridae that infects a member of the genus Trichoderma.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00705-025-06495-z.

Fungal viruses, referred to as mycoviruses, play an important role in fungal biology and ecology. They occur in diverse fungal lineages and are considered to be ubiquitous throughout the fungal kingdom [1–4]. Of the diverse groups of mycoviruses, double-stranded RNA (dsRNA) viruses are among the most extensively studied. Taxonomically, dsRNA mycoviruses are classified into several families, including Partitiviridae, Chrysoviridae, Orthototiviridae, Quadriviridae, Megabirnaviridae, Spinareoviridae, Alternaviridae, and Polymycoviridae (International Committee on Taxonomy of Viruses, http://ictv.global). Fungi belonging to the genus Trichoderma are widely studied because of their ecological significance and potential applications in agriculture and biotechnology [5, 6]. In recent years, the occurrence and characterization of dsRNA mycoviruses have been reported in different Trichoderma isolates using molecular and genomic approaches, revealing diverse genomic organizations and evolutionary relationships [7–18]. So far, members of at least four virus families, including the established families Hypoviridae, Partitiviridae, Fusagraviridae, and the proposed family “Ambiguiviridae”, have been shown to infect Trichoderma spp. Here, we report the identification of a novel mycovirus of the family Alternaviridae, which we have named “Trichoderma harzianum alternavirus 1” (ThAV1), in Trichoderma harzianum strain NFCF419. To the best of our knowledge, this is the first report of the presence of an alternavirus in a member of the genus of Trichoderma.

The fungus T. harzianum NFCF419 was isolated from a sawdust-based cultivation bag of Lentinula edodes showing typical green mold symptoms in Chungcheongbuk-do, Korea [8]. To detect mycoviruses in T. harzianum strain NFCF419, mycelia were cultivated for 5 days on potato dextrose agar (PDA) overlaid with sterilized cellophane, and dsRNA was extracted from fresh biomass using cellulose chromatography [11]. The extracted dsRNA was treated with DNase I and S1 nuclease and then subjected to agarose gel electrophoresis, which revealed a multiple-band pattern indicative of infection by multiple viruses (Fig. 1A). To identify the viral genome sequences, next-generation sequencing (NGS) was performed using the purified dsRNA. Briefly, dsRNA samples were fragmented and reverse transcribed to generate cDNA libraries, which were sequenced on an Illumina HiSeq 2000 platform. Raw sequence data were quality-filtered and adapter-trimmed using Trimmomatic, and de novo assembly was performed using Trinity. This resulted in a total of 5,341 contigs with an average length of 383 nucleotides. The resulting contigs were used for BLASTn and BLASTx searches of the NCBI virus database, which revealed the presence of an alternavirus and a hypovirus, suggesting a mixed infection. The hypovirus was found to be very similar to a previously reported virus [13], but the alternavirus was found to be a novel virus and is the focus of this report. NGS sequencing produced four alternavirus contigs, 3,561, 2,529, 2,570, and 1,471 nt in size. RT-PCR amplification using sequence-specific primer pairs based on the assembled contigs yielded amplicons of the expected sizes, and manual sequencing of the amplicons verified the presence of the viral genome and confirmed the NGS sequence.

Fig. 1.

Profile of dsRNA segments and genomic organization of Trichoderma harzianum alternavirus 1 (ThAV1) from T. harzianum isolate NFCF419. (A) Agarose gel electrophoresis (1% w/v) of ThAV1 dsRNA treated with DNase I and S1 nuclease. Segments are visible as dsRNA1 (~ 3.5 kbp), dsRNA2 and dsRNA3 (~ 2.5 kbp, doublet), and dsRNA4 (~ 1.5 kbp). Lane M, 1-kb DNA ladder. (B) Northern blot analysis of ThAV1. The dsRNAs were separated by electrophoresis under denaturing conditions, blotted onto nylon membranes, and hybridized with ³²P-labeled probes. (C) Systematic diagrams of the dsRNA segments. ORFs are shown as grey boxes, and numbers indicate nucleotide positions

Northern blot analysis using RT-PCR amplicons representing each of the four contigs as probes confirmed the identity of the dsRNA bands at the expected positions in the gel (Fig. 1B), and the terminal sequence of each genome segment was determined using 5’- and 3’-RACE.

The complete nucleotide sequences of the four dsRNA segments were determined to be 3,572 bp (dsRNA1) with a GC content of 53%, 2,552 bp (dsRNA2) with a GC content of 53%, 2,593 bp (dsRNA3) with a GC content of 57%, and 1,484 bp (dsRNA4) with a GC content of 60%, which is consistent with the sizes of the bands observed in agarose gel electrophoresis (Fig. 1A). The nucleotide sequences of the genome segments of the novel alternavirus, which we have named “Trichoderma harzianum alternavirus 1” (ThAV1) were deposited in the GenBank database under the accession numbers PX441381, PX441382, PX441383, and PX441384, respectively. Sequence analysis revealed that each segment contains a single ORF and terminates with a poly(A) tail at the 3′ end (Fig. 1C).

The ORF of dsRNA1 (ORF1) of ThAV1 starts at nucleotide position 72 and ends at position 3,446, and it is predicted to encode a protein of 1,124 amino acids with a calculated molecular weight of 127.8 kDa. A BLASTp search indicated that the deduced amino acid sequence had the highest similarity (59.9% identity) to the RNA-dependent RNA polymerase (RdRP) of Dactylonectria torresensis alternavirus 1 (DtAV1). A multiple alignment of RdRP sequences showed that the RdRp of ThAV1 contains all of the conserved motifs (I-VIII) commonly found in RdRPs. A distinctive feature of alternaviruses, the substitution of glycine (G) by alanine (A) within the metal ion-binding triplet sequence (ADD) in motif VI, was also observed (Fig. 2A). The ADD motif is conserved among alternavirus isolates that are closely related to ThAV1.

Fig. 2.

(A) Multiple sequence alignment of RdRP proteins from ThAV1 and 22 reference alternaviruses, showing the eight conserved motifs (I-VIII) characteristic of RdRPs. (B) Alignment of the 5′-untranslated region (UTR) sequences of ThAV1, with conserved nucleotides shown in dark blue. (C) Phylogenetic tree inferred from the deduced amino acid sequence of the putative RdRP of ThAV1 and those of 22 reference alternaviruses, constructed using the maximum-likelihood (ML) method with 1,000 bootstrap replicates. A representative member of the family Quadriviridae was used as an outgroup. Bootstrap values greater than 70% are shown at the nodes

The ORF of dsRNA2 (ORF2) of ThAV1 extends from nt 66 to nt 2,372, encoding a protein of 768 amino acids with a calculated molecular mass of approximately 84.4 kDa. BLASTp analysis revealed that this protein shared the highest amino acid sequence similarity (36.1% identity) with the putative methyltransferase (MTase) encoded by dsRNA2 of DtAV1 [19], whose counterparts in other alternaviruses have been classified as hypothetical proteins with unknown function [20]. A comparison with the putative MTases of other alternaviruses [19] showed that one highly conserved sequence (GDXPG[T/S][L/F][G/A/S]RXL) is well conserved as “GDHPGSLGRAL” from aa 262 to aa 272, while another conserved sequence (V[V/T]GXDP[K/R]N) is only partially conserved as “SIGIDPLN” from aa 279 to aa 286.

The ORF in dsRNA3 (ORF3) of ThAV1 extends from nt 92 to nt 2,386 and encodes a protein containing 764 aa with a calculated molecular weight of 83.1 kDa. BLASTp analysis revealed that this protein shared the highest amino acid sequence similarity (47.2% identity) with the coat protein encoded by dsRNA3 of DtAV1. Interestingly, the segment of ThAV1 encoding the coat protein is longer than the segment (dsRNA2) encoding the methyltransferase, which has also been observed in the case of Ilyonectria robusta alternavirus 1 (IrAV1) [19].

The ORF of dsRNA4 (ORF4) of ThAV1 extends from nt 218 to nt 1,270 and encodes a protein containing 350 aa with a calculated molecular weight of 37.7 kDa. BLASTp analysis revealed that this protein shared the highest amino acid sequence similarity (29.2% identity) with a hypothetical protein of a different virus, Alternaria alternata alternavirus 1 (AaV1) [21]. The relatively low sequence similarity of this segment to its closest relative reflects the fact that DtAV1, which showed the highest similarity to ThAV1 in the other three segments, consists of only three genome segments and lacks a dsRNA4.

A comparison of the 5’-terminal sequences of the four segments of the ThAV1 genome showed them to be highly diverse but to have the first seven nucleotides 5’-GCCCCGT-3’ in common (Fig. 2B).

Phylogenetic analysis was performed by the maximum-likelihood (ML) method [22] to investigate the evolutionary relationships of ThAV1 to other viruses. The RdRP sequence of ThAV1 was aligned with sequences from reference viruses, using CLUSTALx2 [23], and the alignment was manually inspected and trimmed to remove ambiguous regions. A phylogenetic tree was constructed in MEGA 7 [24] by the ML method with the Jones-Taylor-Thornton (JTT) amino acid substitution model and validated by bootstrap analysis with 1,000 replicates. The RdRP sequence of a virus belonging to the family Quadriviridae was included as an outgroup to root the phylogenetic tree, which showed that ThAV1 clustered with other alternaviruses (Fig. 2C). The 23 alternaviruses included in the analysis formed three clades, and most of them were in clade I, within which ThAV1 and DtAV1 formed a separate subclade.

Based on its genome organization and phylogenetic relationships, ThAV1 can be considered a new member of the family Alternaviridae. Since the first report in 2009, more than 20 viruses have been identified as members of the family Alternaviridae, and many of them infect fungi of the genus Fusarium [20]. To the best of our knowledge, this is the first report of an alternavirus infecting a member of the genus Trichoderma.

Supplementary Material

Below is the link to the electronic supplementary material.

Author contributions

Dae-Hyuk Kim wrote the manuscript. Jeesun Chun and Yo-Han Ko performed the experiments. Dae-Hyuk Kim, Jeesun Chun, and Yo-Han Ko analyzed the data.

Funding

This work was supported by NRF grant no. 2022R1A2C3005906.

Data availability

The genome sequence of the alternavirus has been submitted to the GenBank database.

Declarations

Conflict of interest

The authors declare no conflict of interest.