Prevalence and species diversity of dsRNA mycoviruses from Beauveria bassiana strains in the China's Guniujiang nature.

We investigated the prevalence and species diversity of dsRNA mycoviruses in Beauveria bassiana isolates from the China's Guniujiang Nature Preserve. Among the 28 isolates analyzed, electropherotyping revealed viral infections in 28.6 % (8 out of 28) of the isolates. Metatranscriptomic identification and RT-PCR confirmed the presence of six putative virus species, including two novel species: Beauveria bassiana victorivirus 2 (BbV-2) and Beauveria bassiana bipartite mycovirus 2 (BbBV-2). Four previously characterized mycoviruses were also identified: Beauveria bassiana polymycovirus 4 (BbPmV4), Beauveria bassiana partitivirus 1 (BbPV-1), Beauveria bassiana bipartite mycovirus 1 (BbBV-1), and Beauveria bassiana chrysovirus 2 (BbCV-2). BbPmV4 was found to be the prevailing mycovirus among the infected isolates, and three isolates showed co-infection with both BbPmV4 and BbBV-2. This study enhances our understanding of fungal viral taxonomy and diversity, providing insights into mycovirus infections in B. bassiana populations in China's Guniujiang Nature Preserve. Furthermore, the study on the diversity of B. bassiana viruses lays the foundation for recognizing fungal viruses as potential enhancers of biocontrol agents.

A novel partitivirus orchestrates conidiation, stress response, pathogenicity, and secondary metabolism of the entomopathogenic fungus Metarhizium majus.

Mycoviruses are widely present in all major groups of fungi but those in entomopathogenic Metarhizium spp. remain understudied. In this investigation, a novel double-stranded (ds) RNA virus is isolated from Metarhizium majus and named Metarhizium majus partitivirus 1 (MmPV1). The complete genome sequence of MmPV1 comprises two monocistronic dsRNA segments (dsRNA 1 and dsRNA 2), which encode an RNA-dependent RNA polymerase (RdRp) and a capsid protein (CP), respectively. MmPV1 is classified as a new member of the genus Gammapartitivirus in the family Partitiviridae based on phylogenetic analysis. As compared to an MmPV1-free strain, two isogenic MmPV1-infected single-spore isolates were compromised in terms of conidiation, and tolerance to heat shock and UV-B irradiation, while these phenotypes were accompanied by transcriptional suppression of multiple genes involved in conidiation, heat shock response and DNA damage repair. MmPV1 attenuated fungal virulence since infection resulted in reduced conidiation, hydrophobicity, adhesion, and cuticular penetration. Additionally, secondary metabolites were significantly altered by MmPV1 infection, including reduced production of triterpenoids, and metarhizins A and B, and increased production of nitrogen and phosphorus compounds. However, expression of individual MmPV1 proteins in M. majus had no impact on the host phenotype, suggesting insubstantive links between defective phenotypes and a single viral protein. These findings indicate that MmPV1 infection decreases M. majus fitness to its environment and its insect-pathogenic lifestyle and environment through the orchestration of the host conidiation, stress tolerance, pathogenicity, and secondary metabolism.

Complete genome sequence of a novel victorivirus infecting cicada flower (Cordyceps chanhua).

Cicada flower, Cordyceps chanhua, is a precious edible and medicinal mushroom with uses in both medicine and food in China. In this study, Cordyceps chanhua strain RCEF5995 was found to be coinfected by a previously characterized alternavirus, Cordyceps chanhua alternavirus 1 (CcAV1), and a novel victorivirus, tentatively named "Cordyceps chanhua victorivirus 1" (CcV1). Molecular characterization of CcV1 showed that its complete genome is 5,232 nucleotides long with a GC content of 57.5%. Sequence analysis indicated that CcV1 contains two overlapping open reading frames (ORFs), ORF1 and ORF2, encoding a putative coat protein (CP) of 742 amino acids (aa) and a putative RNA-dependent RNA polymerase (RdRp) of 836 aa, respectively. The termination codon of the CP ORF overlaps with the initiation codon of the RdRp ORF at the tetranucleotide sequence AUGA. Homolog searches, sequence comparisons, and phylogenetic analysis based on deduced amino acid sequences of RdRp indicated that CcV1 is a new member of the genus Victorivirus, family Totiviridae.

A Novel Gammapartitivirus That Causes Changes in Fungal Development and Multi-Stress Tolerance to Important Medicinal Fungus Cordyceps chanhua.

Cicada flower, scientifically named Cordyceps chanhua, is an important and well-known Chinese cordycipitoid medicinal mushroom. Although most mycoviruses seem to induce latent infections, some mycoviruses cause host effects. However, the effects of mycovirus on the fungal development and stress tolerance of C. chanhua remain unknown. In this study, we report a novel mycovirus designated Cordyceps chanhua partitivirus 1 (CchPV1) from C. chanhua isolate RCEF5997. The CchPV1 genome comprises dsRNA 1 and dsRNA 2, 1784 and 1563 bp in length, respectively. Phylogenetic analysis using the aa sequences of RdRp revealed that CchPV1 grouped with members of the genus Gammapartitivirus in the family Partitiviridae. We further co-cultivated on PDA donor strain RCEF5997 and recipient C. chanhua strain RCEF5833 (Vf) for 7 days, and we successfully obtained an isogenic line of strain RCEF5833 with CchPV1 (Vi) through single-spore isolation, along with ISSR marker and dsRNA extraction. The biological comparison revealed that CchPV1 infection slows the growth rate of the host, but increases the conidiation and formation of fruiting bodies of the host. Furthermore, the assessment of fungal tolerance demonstrated that CchPV1 weakens the multi-stress tolerance of the host. Thus, CchPV1 infection cause changes in fungal development and multi-stress tolerance of the host C. chanhua. The findings of this study elucidate the effects of gammapartitivirus on host entomogenous fungi and provide a novel strategy for producing high-quality fruiting bodies of C. chanhua.

Molecular characterization of two novel totiviruses coinfecting the basal fungus Conidiobolus adiaeretus.

A number of viruses have recently been discovered in all major fungal phyla using high-throughput sequencing. However, basal fungi remain among the least-explored organisms with respect to the presence of mycoviruses. In this study, we characterized two mycoviruses coinfecting the basal fungus Conidiobolus adiaeretus, which we have named "Conidiobolus adiaeretus totivirus 1" (CaTV1) and "Conidiobolus adiaeretus totivirus 2" (CaTV2). Due to their similar sizes, the genomic RNAs of these two viruses comigrated as a single band in 1.5% agarose gel electrophoresis but could be distinguished and characterized by next-generation sequencing and RT-PCR. Like those of other totiviruses, the genomes of both CaTV1 and CaTV2 have two discontinuous open reading frames: ORF1 and ORF2, encoding a putative capsid protein and a putative RNA-dependent RNA polymerase (RdRp), respectively. The RdRps of CaTV1 and CaTV2 have 62.73% and 63.76% amino acid sequence identity, respectively, to Wuhan insect virus 26 and have 62.15% amino acid sequence identity to each other. A maximum-likelihood phylogenetic tree based on RdRp amino acid sequences showed that both CaTV1 and CaTV2 clustered in a clade with members of the genus Totivirus. Therefore, we propose that CaTV1 and CaTV2 are two new members of the genus Totivirus in the family Totiviridae.

Molecular characterization of a novel double-stranded RNA virus infecting the entomopathogenic fungus Metarhizium brunneum.

There are four dsRNAs segments present in the entomopathogenic fungus Metarhizium brunneum strain RCEF0766. The genomic segments dsRNA1 and dsRNA3 are of a novel virus, "Metarhizium brunneum bipartite mycovirus 1" (MbBV1), while dsRNA2 and dsRNA4 are the components of the Metarhizium brunneum partitivirus 2 (MbPV2), a member in genus Gammapartitivirus of the family Partitiviridae based on molecular analysis and RT-PCR. This suggests that the strain RCEF0766 was co-infected by two different mycoviruses. The complete genome sequence of MbBV1 was elucidated by high-throughput sequencing and RLM-RACE. MbBV1 consists of two dsRNAs (1987 and 1642 bp) encode open-reading frames (ORFs). The ORF1 in dsRNA 1 encode is a putative RNA-dependent RNA polymerase (RdRp) with the molecular weight of 68.08 kDa, while ORF2 in dsRNA 2 encodes a hypothetical protein with the molecular weight of 33.07 kDa. The deduced proteins of ORF1 and ORF2 have the highest identity to those of Erysiphe necator-associated bipartite virus 1 (76.88% and 65.30%). Based on the amino acid sequence of RdRp, MbBV1 is phylogenetically clustered together with the unassigned mycoviruses and represents a distinct lineage. Our study proposes that MbBV1 is a novel mycovirus with bisegmented dsRNA genomes and should be considered a new member of the unassigned group.

Molecular characterization of a novel alternavirus infecting the entomopathogenic fungus Cordyceps chanhua.

In this study, a novel double-stranded (ds) RNA mycovirus, named Cordyceps chanhua alternavirus 1 (CcAV1), was detected in the entomogenous fungus Cordyceps chanhua in China and characterized. The complete genome of CcAV1 is composed of three dsRNA segments: dsRNA 1 (3,512 bp), dsRNA 2 (2,655 bp), and dsRNA 3 (2,415 bp). Each of the three dsRNAs possesses a single open reading frame (ORF). dsRNA 1 encodes a putative RNA-dependent RNA polymerase (RdRp), and dsRNA 2 and dsRNA 3 encode hypothetical protein 1 (HP 1) and hypothetical protein 2 (HP 2), respectively. The predicted amino acid sequences of the putative RdRp, HP 1, and HP 2 had the highest identity of 66.99%, 49.30%, and 56.91%, respectively, to those of Aspergillus foetidus dsRNA mycovirus. A maximum-likelihood phylogenetic tree based on RdRp amino acid sequences showed that CcAV1 clustered with members of the proposed family "Alternaviridae". Hence, we propose that Cordyceps chanhua alternavirus 1 is a novel member of the proposed family "Alternaviridae".

Molecular characterization of two dsRNAs that could correspond to the genome of a new mycovirus that infects the entomopathogenic fungus Beauveria bassiana.

The entomopathogenic fungus Beauveria bassiana is used worldwide for biological control of insects. Seven dsRNA segments were detected in a single B. bassiana strain, RCEF1446. High-throughput sequencing indicated the presence of three mycoviruses in RCEF1446. Two were identified as the known mycoviruses Beauveria bassiana victorivirus 1 and Beauveria bassiana polymycovirus 1, and the novel mycovirus was designated as "Beauveria bassiana bipartite mycovirus 1" (BbBV1). The complete sequence of the BbBV1 is described here. The mycovirus contains two dsRNA segments. The RNA 1 (dsRNA 4) of BbBV1 is 2,026 bp in length, encoding a RNA-dependent RNA polymerase (RdRp) (68.54 kDa), while the RNA 2 (dsRNA 6) is 1,810 bp in length, encoding a hypothetical protein (35.55 kDa) with unknown function. Moreover, the amino acid sequence of RdRp showed the highest sequence identity of 62.31% to Botryosphaeria dothidea bipartite mycovirus 1. Phylogenetic analysis based on RdRp sequences revealed that BbBV1 represents a distinct lineage of unassigned dsRNA mycoviruses infecting fungi.

Molecular characterization of a novel totivirus infecting the basal fungus Conidiobolus heterosporus.

Mycoviruses are widely distributed in fungi, but only a few mycoviruses have been reported in basal fungi to date. Here, we characterized a novel totivirus isolated from the basal fungus Conidiobolus heterosporus, and we designated this virus as "Conidiobolus heterosporus totivirus 1" (ChTV1). The complete genome of ChTV1 contains two discontinuous open reading frames (ORFs), ORF1 and ORF2, encoding a putative coat protein (CP) and a putative RNA-dependent RNA polymerase (RdRP), respectively. Phylogenetic analysis based on RdRP sequences showed that ChTV1 clustered with members of the genus Totivirus. The RdRP of ChTV1 has 51% sequence identity to that of Trichoderma koningiopsis totivirus 1 (TkTV1), which is the highest among mycoviruses. However, TkTV1 formed a distinct cluster with Wuhan insect virus 27, with 63% RdRP sequence identity, although Wuhan insect virus 27 has not been described, and its host represents a different kingdom. Therefore, we propose that ChTV1 is a new member of the genus Totivirus, family Totiviridae.

A novel gammapartitivirus from the entomopathogenic fungus Metarhizium brunneum.

Here, we report a novel partitivirus infecting Metarhizium brunneum, which was designated "Metarhizium brunneum partitivirus 2" (MbPV2). The complete genome of MbPV2 consists of two segments, dsRNA1 and dsRNA2, with each dsRNA possessing a single open reading frame (ORF). dsRNA1 (1,775 bp) encodes a conserved RNA-dependent RNA polymerase (RdRp) with the highest sequence similarity to Plasmopara viticola associated partitivirus 1 (PvAPV1), while dsRNA2 (1,568 bp) encodes a coat protein (CP) with the highest sequence similarity to Colletotrichum partitivirus 1 (CtParV1). Phylogenetic analysis based on RdRp sequences showed that MbPV2 is a new member of the genus Gammapartitivirus, family Partitiviridae. This is the first report of a gammapartitivirus that infects the entomopathogenic fungus Metarhizium brunneum.

A novel non-segmented double-stranded RNA virus isolated from the basal fungus Conidiobolus sp.

Mycoviruses are widely distributed in a variety of fungal species. However, few viruses have been reported in basal fungi. A novel non-segmented dsRNA virus was isolated from the basal fungus Conidiobolus sp. of the phylum Zoopagomycota, which has been named "Conidiobolus non-segmented RNA virus 1" (CNRV1). The complete genome sequence of CNRV1 was determined by dsRNA extraction, next-generation sequencing, and RACE. The genome of CNRV1 dsRNA is 3,092 bp in length and contains two open reading frames (ORFs) predicted to encode a subgenomic protein 1 (sgP1) and a putative RNA-dependent RNA polymerase (RdRp). Multiple sequence alignment and phylogenetic analysis based on RdRp sequences from selected dsRNA viruses showed that CNRV1 shared 31.9% sequence identity with Nigrospora oryzae unassigned RNA virus 1 (NoNRV1) and clustered with NoNRV1 and four other mycoviruses. These viruses are unassigned and distant from members of the family Partitiviridae, although they were previously considered partitivirus-like viruses. Thus, CNRV1 is a novel member of proposed genus "Unirnavirus", and is the first dsRNA sequence reported from a member of the phylum Zoopagomycota. This study extends our knowledge about mycoviruses in basal fungi.

Molecular characterization of a new partitivirus, MbPV1, isolated from the entomopathogenic fungus Metarhizium brunneum in China.

Mycoviruses from Metarhizium anisopliae have been extensively studied, but their sequences have yet to be deposited in the NCBI database. In the present study, we characterized a new partitivirus obtained from the entomogenous fungus Metarhizium brunneum, named "Metarhizium brunneum partitivirus 1" (MbPV1). The complete genome of MbPV1, determined by metagenomic sequencing, RT-PCR, and RACE, comprised two dsRNA segments of 1,829 bp and 1,720 bp, respectively. Both dsRNAs contained a single open reading frame (ORF), encoding a putative RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively. The sequences of the RdRp and CP showed the highest similarity (61.4% and 44.4% identity, respectively) to those of Colletotrichum eremochloae partitivirus 1 (CePV1), which were obtained from the NCBI database. A phylogenetic tree based on the RdRp sequence showed that MbPV1 clustered with members of the proposed genus "Epsilonpartitivirus", belonging to family Partitiviridae. Here, we propose that MbPV1 is a member of a new species of the proposed genus "Epsilonpartitivirus". This is the first sequence data report of a new mycovirus from a member of the genus Metarhizium.

Complete genome sequence of a novel partitivirus from the entomogenous fungus Beauveria bassiana in China.

In this study, we report a novel double-stranded RNA (dsRNA) virus, Beauveria bassiana partitivirus 3 (BbPV-3), derived from the entomogenous fungus Beauveria bassiana isolate RCEF5853 from China. The genome of BbPV-3, whose sequence was determined by metagenomic sequencing, RT-PCR, and RACE cloning, comprises two dsRNA genome segments that are 1,856 and 1,719 bp long. The first segment contains a single ORF (ORF-1) encoding a 584-amino-acid-long protein (66.05 kDa) with a conserved RNA-dependent RNA polymerase (RdRp) motif. The second segment also has a single ORF (ORF-2) encoding a 500-amino-acid-long coat protein (CP) (55.9 kDa). The CP and RdRp sequences showed highest identity of 43.4% and 60.2%, respectively, to those of Colletotrichum eremochloae partitivirus 1. Phylogenetic analysis of the RdRp domain of the polyprotein revealed that BbPV-3 grouped together with the members of the genus Epsilonpartitivirus. Hence, we proposed that Beauveria bassiana partitivirus 3 is a novel member of the proposed genus Epsilonpartitivirus.