Sequencing of the mycovirus genome
From the 150 Trichoderma strains, we identified a strain designated HB40525 (or 525), containing typical dsRNAs of mycoviruses. Electrophoresis showed that the putative mycovirus fragment was approximately 3 kb in length (Fig. 1). Using next-generation sequencing (NGS) analysis, the contig-36 was determined to have a size of approximately 3 kb and to show high similarity to three mycoviruses: Alternaria longipes dsRNA virus 1 [25], Penicillium janczewskii Beauveria bassiana-like virus 1 [26], and Beauveria bassiana RNA virus 1 [27]. The strains exhibited sequence homology of 63.50% to Alternaria longipes dsRNA virus 1, YP_009052469.1 [25], 57.30% to Penicillium janczewskii Beauveria bassiana-like virus 1, ALO50135.1 [26] and 57% to Beauveria bassiana RNA virus 1, AKC57301.1 [27] (Additional file 1: Table S2). Using the sequence of contig-36, primers for RT-PCR were designed, and RT-PCR was performed. The cDNA sequences of the mycovirus without the 5′ and 3′ termini were verified. The 5′ and 3′ terminal sequences of the cDNAs were obtained by the classic 5′ RACE and 3′ RACE cloning methods (Fig. 1, Additional file 1: Table S3 and Figure S1). The results indicated that the 3′ terminus of the virus contained 15 poly(A) structures. Additional structures within the 5′ and 3′ termini were evaluated using RNA structure 3.0 (Additional file 1: Figure S2). The cDNA sequences and RACE sequences were assembled to obtain the complete sequence of the virus using DNAMAN software. The resulting mycovirus genome was 3160 bp (including the poly(A) tail) and was submitted to GenBank under accession number MH155602.
Structure of the mycovirus genome
In the genome sequence, the G + C content was 51.98%. The coding strand of the dsRNA exhibited two ORFs on the negative strand, which constituted the coding strand of the mycovirus. ORF-A (residues 1857–109) encoded an RNA-dependent RNA polymerase (RdRP), which was a protein of 582 amino acids with a molecular weight of approximately 66 kDa. ORF-B (residues 3047–1908) encoded a putative protein of 379 amino acids with a molecular weight of approximately 41 kDa. The positive strand harboured ORF C (residues 1076–1370), presumed to encode a hypothetical protein containing 94 amino acids with a poly(A) structure at the 3′ terminus (Fig. 2, Additional file 1: Figure S2).
Phylogenetic analysis of the mycovirus and its taxonomic status
An NCBI BLASTN search identified five closely related fungal viruses with amino acid sequences similar to that of ORF-A: Alternaria longipes dsRNA virus 1 [25] (AlRV1, YP_009052469.1, homologous at 63% sequence identity), Penicillium janczewskii Beauveria bassiana-like virus 1 [26] (PjBlV1, ALO50135.1, homologous at 57% sequence identity); Beauveria bassiana RNA virus 1 [28] (BbNV-1, YP_009154711.1 homologous at 57% sequence identity); Beauveria bassiana RNA virus 1 [27] (BbV1-A24, AKC57301.1, homologous at 57% sequence identity); and Colletotrichum higginsianum non-segmented dsRNA virus 1 [29] (ChNRV1, YP_009177217.1, homologous at 55% sequence identity). The phylogenetic tree with the best model indicated that these fungal mycoviruses belonged to different families (Fusagraviridae, Megabirnaviridae, Totiviridae, Chrysoviridae), and the mycovirus of strain 525 belonged to the unclassified group structure [30] (Fig. 3a and Additional file 1: Table S4).
Based on the amino acid sequence of ORF-B, we found five closely related fungal viruses using NCBI BLASTn: AlRV1 (YP_009052468.1 homologous at 41% sequence identity); BbNV-1 (YP_009154710.1 homologous at 42% sequence identity); BbV1-A24 (AKC57300.1, homologous at 44% sequence identity); PjBlV1 (ALO50134.1, homologous at 44% sequence identity); and ChNRV1 (YP_009177216.1, homologous at 43% sequence identity). When all of these sequences were analysed using the best model, LG + G + I + F, ORF-B was grouped with the five mycoviruses with high similarity. The BbNV-1 protein is a putative coat protein, so ORF-B of strain 525 was considered to also encode a coat protein (Fig. 3b and Additional file 1: Table S5).
From the BLAST results for ORF-C based on the amino acid sequences from the NCBI database, only one hypothetical protein from Mycolicibacterium conceptionense (WP_076212170.1) was discovered, with 34% identity.
For the sake of completeness, the alignment that resulted from concatenating the aligned RdRP and coat protein sequences was also used to construct a single maximum likelihood phylogenetic tree (using the rtREV+G + I + F model) (Fig. 3c and Additional file 1: Table S6). The results were identical to the phylogenetic trees inferred using each protein independently. We concluded that the mycovirus from T. harzianum strain 525 was novel and unclassified, and we named it Trichoderma harzianum mycovirus 1 (ThMV1).
Biological effects of ThMV1 on Trichoderma harzianum 525
The elimination of the mycovirus from Trichoderma strain 525 was successfully performed using ribavirin and protoplasting/regeneration. RT-PCR of T525 and T525-F was performed to verify the elimination of ThMV1. The results showed that T525 exhibited a fragment length of 2997 bp, but T525-F did not contain the same fragment (Figs. 1 and 4a). No dsRNA from T525-F was detected by Northern blot analysis using a DIG-labelled probe, confirming that the mycovirus was successfully removed from T525-F (Fig. 4b).
When the growth rate of the two strains cultured on different media was measured, we found no statistically significant differences between them (Fig. 5d and Additional file 1: Tables S7, S8, S9, and S10, ANOVA P = 0.095), although on the 8th day, the hyphae of T525 had already covered the entire plate, while the hyphae of the T525-F had not yet done so (Fig. 5c). There were differences between the two growth media, however, with the hyphae growing almost twice as fast on PDA as on CZA (Fig. 5a and b and Additional file 1: Tables S7, S8, S9, and S10, ANOVA P < 0.001). No significant strain-by-media interaction was observed (Fig. 5d and Additional file 1: Table S9, ANOVA P = 0.389), confirming that the similarity of growth rates between the two strains was independent of the medium. Because the CMD medium was thick and opaque, we could not measure the length of the hyphae accurately, but T525 grew more vigorously and densely than T525-F (Fig. 5b).
In contrast to the lack of difference between T525 and T525-F in terms of growth rates, the biomass produced by T525 was 13.25% greater than that of T525-F (Fig. 5e), a difference that was statistically significant (two-sample t-test: t = 2.117, 38 d.f., P = 0.041) (Additional file 1: Tables S8 and S9). Thus, the presence of the mycovirus had a beneficial effect on the production of mycelia.
To evaluate biocontrol capabilities, we tested the antagonism of T525 and T525-F against three pathogenic fungi (F. oxysporum f.sp. cucumebrium Owen, B. cinerea and F. oxysporum f. sp. vasinfectum). There were no obvious differences in vitro (Additional file 1: Figure S2), but the control effectiveness towards cucumber wilt disease caused by F. oxysporum f.sp. cucumebrium Owen was significantly different in vivo. While the cucumber seedlings in the T1, T3, and T4 treatments all grew well, little difference existed in the observed growth trend; all of the cucumber seedlings of T3 grew more vigorously and exhibited more fibrous roots than T1 and grew higher than T4 by the 12th day (Fig. 6). Moreover, on the 24th day, the cucumber seedlings of T3 were all larger than those of T1 and T4, with larger true leaves and more fibrous roots (Fig. 7), which indicated that T525 had the potential to function to improve cucumber growth. The T2 seedlings infected only with F. oxysporum f. sp. cucumebrium Owen were all withered 7 days after inoculation, which indicated that the cucumber species exhibited no resistance to this pathogen. However, the growth trends of T6 were all better than those of T2 and T5. T2 showed severe disease symptoms, whereas the growth trend of T5 only showed the initial stage of withering, with all leaves beginning to turn yellow (Fig. 6 and Additional file 1: Figure S3).
These results together indicate that T525 possesses the capacity to promote plant growth, while T525-F could improve the pathogen resistance of plants. The milder disease symptoms of cucumber seedlings treated with T525-F implied that ThMV1 interacted with its host, T525, and decreased the host’s biocontrol efficiency against plant disease. Plants treated with T525 (T3) appeared to grow significantly better than those that did not receive this treatment (T1) or were treated with T525-F (T4), and T1 was observed to promote better growth than T4.