A novel double-stranded RNA mycovirus isolated from Trichoderma harzianum
Trichoderma spp. are used extensively in agriculture as biological control agents to prevent soil-borne plant diseases. In recent years, mycoviruses from fungi have attracted increasing attention due to their effects on their hosts, but Trichoderma mycoviruses have not been the subject of extensive study. We sought to discover novel mycoviruses from Trichoderma spp. and to determine the effects of the biocontrol function of Trichoderma spp.
Mycoviruses were screened by dsRNA extraction and metagenomic analysis. RT-PCR, 5′ RACE, and 3′ RACE were used to obtain the genome sequence. MEGA software was used to classify the new mycovirus. The effects of the identified mycovirus on the biological properties of the host strain 525 were evaluated using cucumber plants and Fusarium oxysporum f. sp. cucumerinum.
A novel mycovirus, Trichoderma harzianum mycovirus 1 (ThMV1) (accession number MH155602), was discovered in Trichoderma harzianum strain 525, a soil-borne fungus collected from Inner Mongolia, China. The mycovirus exhibited a double-stranded RNA (dsRNA) genome with a complete genome sequence of 3160 base pairs and two open reading frames (ORFs) on the negative strand. Phylogenetic analysis indicated that it belongs to an unclassified family of dsRNA mycoviruses. The removal of ThMV1 from the host 525 strain reduced host biomass production and improved the biocontrol capability of the host for Fusarium oxysporum f. sp. cucumerinum. At same time, the presence of ThMV1 improved the growth of cucumber.
ThMV1 is a new unclassified mycovirus found in T. harzianum. It not only affects the phenotype of the host strain but also reduces its biocontrol function, which sheds light on the interaction between the mycovirus and Trichoderma spp.
KeywordsMycovirus Trichoderma harzianum dsRNA Trichoderma harzianum mycovirus 1
Analysis of variance
Basic Local Alignment Search Tool
Corn Meal Dextrose Agar
- et al.
Glass test tube
Molecular evolutionary genetics analysis
Murashige and Skoog
National Center for Biotechnology Information
Open reading frame
Polymerase chain reaction
Potato dextrose agar
Parts per million
Rapid amplification of cDNA ends
RNA-dependent RNA polymerase
Revolutions per minute
Ribosomal RNA (rRNA);
Reverse-transcription polymerase chain reaction
The 525 strain with mycovirus
The 525-F strain without mycovirus
Terminal deoxynucleotidyl transferase
Mycoviruses are widespread viruses that infect filamentous fungi and yeasts, and most do not cause their hosts to exhibit obvious symptoms [1, 2]. The majority of mycoviruses exhibit a double-stranded RNA (dsRNA) genome, which is diagnostic. However, a few mycoviruses exhibit single-stranded RNA (ssRNA), double-stranded DNA (dsDNA), or single-stranded DNA (ssDNA) genomes . Based on the mode of replication and the type of genome, the International Committee on Taxonomy of Viruses has divided all currently known mycoviruses into 16 families and an unclassified group. The 16 families include seven dsRNA virus families, five positive-sense ssRNA virus families, two reverse-transcription virus families (+ssRNA), one negative-sense ssRNA virus family, and one positive-sense ssDNA virus family . The taxonomic status of approximately 20% of fungal viruses has yet to be determined [5, 6].
Mycoviruses are transferred from one fungal isolate to another through hyphal fusion (anastomosis) or from parent to offspring via spores (mostly via fusion of asexual spores but also sexual spores). Some mycoviruses can be eliminated in sexual spores. For example, ssRNA and dsRNA viruses in Basidiomycetes can be transmitted by basidiospores, but they can also be removed from the basidiospores produced by Helicobasidium mompa . In some cases, they can be virulent to their hosts, but most mycovirus infections are asymptomatic .
In recent years, mycoviruses have drawn increased attention for the effects they can have on their host virulence ; some can significantly reduce virulence (e.g., the dsRNA virus DK21 of Fusarium graminearum ), while others can significantly increase it (e.g., the dsRNA mycovirus RsRV2 found in the rice sheath blight isolate D122 strain ). These findings have spurred the development of hypovirulence-associated mycoviruses for use as biocontrol agents [12, 13, 14].
Mycoviruses exhibit two main mechanisms that facilitate their function as biocontrol agents of plant pathogenic fungi: first, they can cause the host to become a low-virulence strain; second, the metabolites induced by the mycovirus can increase the pathogenicity of the host [15, 16, 17]. Although some low-virulence mycoviruses have been found in phytopathogenic fungi, most are still in the research stage examining their potential as biocontrol agents. The most successful mycovirus biocontrol agent identified to date has been Cryphonectria parasitica hypovirus 1 (CHV1), which was the first low-virulence mycovirus employed to both prevent and treat disease .
Trichoderma spp. are anamorphic soil-borne filamentous fungi classified as belonging to phylum Ascomycota, class Sordariomycetes, order Hypocreales, and family Hypocreales. While they are well studied , their mycoviruses have not been sufficiently investigated .
Here, we chose 150 strains of Trichoderma spp. isolated from Xinjiang, Inner Mongolia, Jilin, and Heilongjiang Provinces, China, to screen mycoviruses using metagenomics and molecular biology methods. One mycovirus was recovered, and its genome and molecular characteristics are described along with its biological properties.
One hundred and fifty Trichoderma spp. strains were isolated from the forests and grasslands of Xinjiang, Inner Mongolia, Jilin, and Heilongjiang Provinces, China, in 2014–2016 (Additional file 1: Table S1). All fungal strains were cultured on potato dextrose agar (PDA) plates for 7 days at 28 °C.
Extraction and purification of dsRNA
dsRNA was extracted from the mycelia using CF-11 cellulose column chromatography . To isolate dsRNA, the mycelia were grown for 7–10 days on cellophane membranes placed on top of the PDA medium in Petri dishes. Following harvest, the mycelia were stored at − 80 °C. dsRNA was extracted according to a previously described method  and then treated with RNase-free DNase Ι (TaKaRa, Dalian, China) and S1 Nuclease (TaKaRa, Dalian, China) following the manufacturer’s instructions to remove any DNA and ssRNA contamination. Finally, electrophoresis in a 1.5% agarose gel was performed, and t the dsRNA was detected by UV transillumination.
dsRNA was extracted and sequenced using high-throughput sequencing (Illumina HiSeq 2000/2500) at the Biotechnology Corporation (Biotechnology Corporation, Shanghai, China). Briefly, purified dsRNA was first assessed for quality. Ribosomal RNA (rRNA) was removed, and the dsRNA was fragmented. Next, first-strand complementary DNAs (cDNAs) were synthesized, followed by second-strand cDNAs. The complete sample sequencing library was obtained, and the full library was sequenced. The sequencing data were then analysed; unigenes were annotated with RefSeq; and non-redundant proteins according to the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) were searched to screen mycovirus-related sequences.
Cloning and sequencing of the mycovirus
cDNA of the dsRNA were obtained by reverse-transcription polymerase chain reaction (RT-PCR) based on the contig obtained from metagenomics analysis (forward primer: 5′ TCGATGTACGGATTCTCGTGC 3′; reverse primer: 5′ TGTCATCATCGTCTTCAGCCC 3′; fragment length: 2997 bp). Having obtained most of the virus sequence, the 5′- and the 3′-termini were cloned using the rapid amplification of cDNA ends (RACE) method. The 5′-end was obtained by the classic 5′-RACE cloning method  (Additional file 1: Table S3). A poly(A) tail was added to the 3′-end of the cDNA using terminal deoxynucleotidyl transferase (TDT). Linker primers and specific primers were used to perform nested PCR amplification. The 3′-end of the virus was obtained through the 3′-RACE cloning method  (Additional file 1: Table S3). Using the QT hybrid primer software, specific primers were designed for nested PCR amplification.
The RT-PCR products were purified using a gel extraction kit (Tiangen, Germany), ligated to the PMD18-T vector, and submitted for sequencing. Using DNAMAN software, the cDNA clone sequence of the virus and the RACE clone sequence were assembled to form a complete viral genome sequence.
Complete genome sequence and phylogenetic analysis
Mycovirus ORFs were identified by the ORF finder program from the NCBI website (https://www.ncbi.nlm.nih.gov/orffinder/). The mycovirus sequence was searched using the NCBI BLAST program to identify similar mycoviruses for phylogenetic analysis. The phylogenetic tree was constructed using the maximum likelihood method for the 29 chosen mycovirus sequences of RdRP, 22 mycovirus sequences of hypothetical proteins or CPs, and 22 RdRP+ hypothetical proteins or CPs using MEGA 6.0 software based on the best models of nucleotide substitution: K2 + G + I, LG + G + I + F, and rtREV+G + I + F (Additional file 1: Tables S4, S5 and S6).
Effect of the mycovirus on the biological properties of Trichoderma strain 525
Tthe inhibitory function of ribavirin was used to establish an analysis system for eliminating mycovirus from Trichoderma strain 525 by protoplasting/regeneration. The enzymatic hydrolysate used for the preparation of protoplasts of Trichoderma strain 525 was Trichoderma lysing enzyme (BingDa Biotechnology Company, Beijing). After enzymatic lysis, a 200 μL aliquot of protoplasts was applied to regeneration medium that contained ribavirin (100 μM) (BingDa Biotechnology Company, Beijing), followed by culture at 28 °C for 2 days. Single colonies were then picked and transferred to PDA medium that contained ribavirin at 100 μM, followed by culture for 7 days. To check if the mycovirus was present, dsRNA was extracted , after which RT-PCR and northern blotting were used to reconfirm whether dsRNA had been eliminated from the Trichoderma strain. A digoxigenin (DIG)-labelled probe was synthetized using the PCR DIG Probe Synthesis Kit (Roche Diagnostics, USA) with the following RT-PCR primers: forward primer: 5′ TCGATGTACGGATTCTCGTGC 3′; reverse primer: 5′ TGTCATCATCGTCTTCAGCCC 3′. The length of the RT-PCR fragment was 2997 bp.
The biological functions of the 525 strain with the mycovirus (T525) and the strain without the mycovirus (T525-F) were evaluated. Both strains were cultured in PDA, corn meal dextrose agar (CMD) medium, and Czapek–Dox (CZA) medium at 28 °C for 8 days. The length of hyphal growth was measured every 24 h, with thirty replicates in each case. To obtain the differences between the strains, the hyphal growth rates of the two strains were compared with each other. The T525 and T525-F strains were inoculated into PD liquid medium and incubated at 28 °C with 180 rpm shaking for 2 days. The dry weights of the mycelia in each bottle were then determined, and the biomasses of the two strains were compared, with twenty replicates in each case.
To evaluate biocontrol capabilities in vitro, antagonistic experiments involving T525 and T525-F were carried out. The three pathogens Fusarium oxysporum f.sp. cucumebrium Owen, Botrytis cinerea, and Fusarium oxysporum f. sp. vasinfectum were chosen for dual culture to analyse the characteristics of the antagonism of T525 and T525-F.
The biocontrol effect of T525 and T525-F against F. oxysporum f. sp. cucumerinum was further explored in vivo in cucumber plants. Six treatments were designed (Additional file 1: Figure S4): T1, in which cucumber seeds were planted in plates with sterilized water and incubated at 28 °C until the 6th day, after which the seedlings were transferred to glass test tubes (GTTs) containing 10 mL of 1/8 MS; T2, in which cucumber seeds were planted in plates with sterilized water and then incubated at 28 °C until the 4th day, after which the seedlings were exposed with 1 × 107 CFU/mL spores of F. oxysporum f. sp. cucumerinum by dipping, cultured for one day, and then transferred to GTTs containing 10 mL of 1/8 MS on the 6th day; T3, in which the cucumber seeds were only treated by dipping with 1 × 107 CFU/mL spores of T525, then transferred to plates with sterilized water and incubated at 28 °C until the 6th day, after which they were transferred to GTTs containing 10 mL of 1/8 MS; T4, in which the cucumber seeds were only treated by dipping with 1 × 107 CFU/mL spores of T525-F, then transferred to plates with sterilized water and incubated at 28 °C until the 6th day, after which they were transferred to GTTs containing 10 mL of 1/8 MS; T5, in which the cucumber seeds were treated by dipping with 1 × 107 CFU/mL spores of T525, then transferred to plates with sterilized water and incubated at 28 °C. On the 4th day, the germinated seedlings were treated by dipping with 1 × 107 CFU/mL spores of F. oxysporum f. sp. cucumerinum and cultured for one day until the 6th day, when they were transferred to GTTs containing 10 mL of 1/8 MS; T6, in which the cucumber seeds were treated by dipping with 1 × 107 CFU/mL spores of T525, then transferred to plates with sterilized water and incubated at 28 °C. On the 4th day, the germinated seedlings were treated by dipping with 1 × 107 CFU/mL spores of F. oxysporum f. sp. cucumerinum and cultured for one day, then transferred to GTTs containing 10 mL of 1/8 MS on the 6th day. On the 12th day, all treatments were observed. Every treatment was carried out in three repeat experiments with thirty replicates in total. For treatments T1, T3, and T4, plant growth continued to be observed until the 24th day. The plates for all treatments were filled every day with sterilized water, and GTTs were filled to a constant volume of 10 mL. Every five plants were photographed for every treatment.
Sequencing of the mycovirus genome
Structure of the mycovirus genome
Phylogenetic analysis of the mycovirus and its taxonomic status
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
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.
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.
This study describes a new mycovirus found in T. harzianum strain 525, which was isolated from grassland soils in Inner Mongolia, China. Through sequence alignment, the mycovirus was found to exhibit high sequence homology with Alternaria longipes dsRNA virus 1, Penicillium janczewskii Beauveria bassiana-like virus 1, Beauveria bassiana RNA virus 1, Beauveria bassiana RNA virus 1, and Colletotrichum higginsianum non-segmented dsRNA virus 1, with sequence identities of 44–50.5% (Additional file 1: Table S6). Based on phylogenetic analysis of its amino acid sequence, this mycovirus was identified as a new unclassified type of mycovirus.
To date, only three isolates of mycoviruses have been recovered from Trichoderma spp. Jom-in and Akarapisan (2009) isolated two mycoviruses with sizes of 0.7 kb and 1.1 kb from Trichoderma spp., although their taxonomic status was not analysed or classified . Yun et al. (2016) noted that mycoviruses of Lentinula edodes are widespread in fungi . They isolated 32 strains containing dsRNA from 315 Trichoderma strains, which were divided into 15 groups according to dsRNA electrophoresis patterns. Although colony morphological characteristics differed among the groups , accurate mycovirus sequences were not obtained. Lee et al. (2017) also isolated an unclassified mycovirus from T. atroviride referred to as Trichoderma atroviride mycovirus 1 (TaMV1) .
Previous studies on the mycoviruses of fungal pathogens have shown that they can affect the virulence of host fungi. For instance, Li et al. (2015) found that Fusarium graminearum Hypovirus 2 (FgHV2/JS16) could significantly reduce the pathogenicity of Fusarium graminearum by inhibiting the production of deoxynivalenol (DON) toxin . Zhang et al. (2014) found that the dsRNA mycovirus Rhizoctonia solani RNA virus 2 (RsRV2) from the rice sheath blight isolate D122 strain, which belongs to the family Paritiviridae, increased the virulence of host fungus towards plants . Jom-in et al. (2009) tried to remove the mycovirus from TM10 and TM20 hosts but was unable to obtain Trichoderma strain-free mycovirus . Jom-in investigated the antagonistic activity of Trichoderma spp. without mycoviruses and indirectly verified that TM10 and TM20 mycoviruses decreased the antagonistic action of Trichoderma spp. . In our study, the mycovirus of T525 was successfully eliminated, and ThMV1 did not affect the antagonistic activities of the host in vitro, although it obviously decreased the biocontrol efficiency of T525 against F. oxysporum f.sp. cucumebrium Owen in vivo. ThMV1 also exhibited the potential to induce plant growth. This study is the first to report the biocontrol traits of a mycovirus from Trichoderma spp., highlighting the interactions between T. harzianum, F. oxysporum f. sp. cucumebrium Owen and ThMV1.
ThMV1 is the first reported mycovirus that not only affects the biomass of the host strain but also reduces the biocontrol function of Trichoderma and promotes the growth of plants. This work provides insight into how to locate and isolate mycoviruses that could potentially affect the biocontrol function of Trichoderma. The interaction mechanisms among the mycovirus, Trichoderma, pathogen, and plant will be the focus of future investigations.
We are grateful to Prof. Santiago F. Elena (I2SysBio-CSIC, Valencia, Spain) for many helpful suggestions during this research.
CL and ML performed the experiments; ETR, JM and JZ isolated Trichoderma spp., CL and BW analysed the data and wrote the manuscript; BW and XJ designed the study. All authors read and approved the final manuscript.
This work was supported by the National Key Research and Development Plan (Chemical Fertilizer and Pesticide Reducing Efficiency Synergistic Technology Research and Development): Science and Technology Foundation of the Ministry of Science and Technology of China (2014FY120900–4) granted to Xiliang Jiang; Natural Science Foundation of Beijing, China: Exploration of Mycovirus of Trichoderma and Their Effects on the Host Biology (No. 6192022) granted to Beilei Wu; Demonstration of Comprehensive Prevention and Control Technology of Non-point Source Pollution in Main Vegetable-Producing Areas of Huang Huai Hai (SQ2018YFD080026) granted to Beilei Wu.
Ethics approval and consent to participate
This article does not contain any studies with human participants performed by any of the authors.
Consent for publication
Informed consent was obtained from all participants included in the study.
The authors declare that they have no competing interests.
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