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.

The ARP2/3 complex, acting cooperatively with Class I formins, modulates penetration resistance in Arabidopsis against powdery mildew invasion.

The actin cytoskeleton regulates an array of diverse cellular activities that support the establishment of plant-microbe interactions and plays a critical role in the execution of plant immunity. However, molecular and cellular mechanisms regulating the assembly and rearrangement of actin filaments (AFs) at plant-pathogen interaction sites remain largely elusive. Here, using live-cell imaging, we show that one of the earliest cellular responses in Arabidopsis thaliana upon powdery mildew attack is the formation of patch-like AF structures beneath fungal invasion sites. The AFs constituting actin patches undergo rapid turnover, which is regulated by the actin-related protein (ARP)2/3 complex and its activator, the WAVE/SCAR regulatory complex (W/SRC). The focal accumulation of phosphatidylinositol-4,5-bisphosphate at fungal penetration sites appears to be a crucial upstream modulator of the W/SRC-ARP2/3 pathway-mediated actin patch formation. Knockout of W/SRC-ARP2/3 pathway subunits partially compromised penetration resistance with impaired endocytic recycling of the defense-associated t-SNARE protein PEN1 and its deposition into apoplastic papillae. Simultaneously knocking out ARP3 and knocking down the Class I formin (AtFH1) abolished actin patch formation, severely impaired the deposition of cell wall appositions, and promoted powdery mildew entry into host cells. Our results demonstrate that the ARP2/3 complex and formins, two actin-nucleating systems, act cooperatively and contribute to Arabidopsis penetration resistance to fungal invasion.

Co-infection of Aspergillus ochraceopetaliformis strain RCEF7483 by a novel chrysovirus and a known partitivirus.

An airborne microflora isolate, Aspergillus ochraceopetaliformis RCEF7483, was found to harbor seven dsRNA elements, indicating co-infection with a novel chrysovirus and a known partitivirus. Sequence analysis and RT-PCR confirmed dsRNA5-7 as components of Aspergillus ochraceous virus (AOV), a member of the Partitiviridae family. In light of its distinct host, we have designated it Aspergillus ochraceopetaliformis partitivirus 1 (AoPV1). The dsRNA segments, named dsRNA1-4, with lengths of 3706 bp, 3410 bp, 3190 bp, and 3158 bp, respectively, constitute the genome of a novel chrysovirus designated Aspergillus ochraceopetaliformis chrysovirus 1 (AoCV1). The dsRNA1-4 segments contain five open-reading frames (ORF1-5). Specifically, ORF1 encodes a putative RNA-dependent RNA polymerase (RdRp) with a length of 1112 amino acids, and ORF2 encodes a putative coat protein (CP) spanning 976 amino acids. Additionally, ORF3-5 encode hypothetical proteins (HP1, HP2, and HP3) with lengths of 108, 843, and 914 amino acids, respectively. Comparative analysis revealed the highest similarity of dsRNA1-4 with corresponding proteins in Aspergillus terreus chrysovirus 1 (AtCV1) (RdRp, 66.58%; CP, 51.02%; HP2, 61.80%; and HP3, 41.30%). Due to falling below the threshold for a new species in the Chrysoviridae, we propose that dsRNA1-4 in A. ochraceopetaliformis strain RCEF7483 constitute the novel chrysovirus AoCV1. Moreover, phylogenetic analysis using RdRp amino acid sequences placed AoCV1 within the Alphachrysovirus genus of the Chrysoviridae family, clustering with AtCV1 and other alphachrysoviruses. Our study contributes to the understanding of mycoviruses in A. ochraceopetaliformis and expands our knowledge of the diversity and evolution of chrysoviruses in fungal hosts.

Complete genome sequence of a novel gammapartitivirus from Penicillium oxalicum RCEF7482.

Penicillium oxalicum, an important biocontrol fungus in China, has been a subject of extensive study due to its role in combating various pathogenic fungi. Despite the prevalence of mycoviruses with double-stranded (ds) RNA genomes in filamentous fungi, there has been no screening of mycoviruses in P. oxalicum. In this report, we describe the identification and characterization of a novel dsRNA virus isolated from P. oxalicum, designated as "Penicillium oxalicum partitivirus 1" (PoPV1). The genome of PoPV1 consists of two dsRNA segments, dsRNA1 (1,770 bp) and dsRNA2 (1,584 bp), each containing a single open reading frame (ORF): ORF1 and ORF2. Comparative analysis revealed that the RdRp and CP amino acid sequences of PoPV1 share the highest identity (89.18% and 73.97%, respectively) with those of Penicillium aurantiogriseum partitivirus 1 (PaPV1). Motif analysis based on RdRp amino acid sequences places PoPV1 in the genus Gammapartitivirus within the family Partitiviridae, with a distinctive motif VI (R/KV/ILGDD). Phylogenetic analysis further established a close relationship of PoPV1 to PaPV1, forming a unique clade among the gammapartitiviruses. Consequently, we propose that Penicillium oxalicum partitivirus 1 represents a new species in the genus Gammapartitivirus. This is the first report of a dsRNA virus in P. oxalicum.

A novel betapartitivirus isolated from Cordyceps militaris, an edible-medicinal mushroom.

In this study, we identified a novel partitivirus, named "Cordyceps militaris partitivirus 1" (CmPV1), in Cordyceps militaris strain RCEF7506. The complete genome of CmPV1 comprises two segments, dsRNA1 and dsRNA2, each encoding a single protein. dsRNA1 (2,206 bp) encodes an RNA-dependent RNA polymerase (RdRp), and dsRNA2 (2,256 bp) encodes a coat protein (CP). Sequence analysis revealed that dsRNA1 has the highest similarity to that of Bipolaris maydis partitivirus 2 (BmPV2), whereas dsRNA2 shows the highest similarity to human blood-associated partitivirus (HuBPV). Phylogenetic analysis based on RdRp sequences suggests that CmPV1 is a new member of the genus Betapartitivirus of the family Partitiviridae. This is the first documentation of a betapartitivirus infecting the entomopathogenic fungus C. militaris.

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".

A novel polymycovirus infecting the entomopathogenic fungus Metarhizium brunneum.

In this study, at least four distinct double-stranded RNA viruses, including two partitiviruses, one bipartite virus, and a novel polymycovirus, were detected in the entomopathogenic fungus Metarhizium brunneum strain RCEF0736. We describe the characterization of one of these viruses, a novel polymycovirus, which we have named "Metarhizium brunneum polymycovirus 1" (MbPmV1). The genome of MbPmV1 has four dsRNA segments, ranging from 1153 to 2421 bp in length. dsRNA1, 2, and 3 of MbPmV1 each contain a single large open reading frame (ORF), while dsRNA4 has two ORFs. BLASTp analysis indicated that dsRNA1, 2, 3, and 4 of MbPmV1 have a high degree of similarity to the putative RdRp (59.45%), serine protease (44.22%), putative methyltransferase (48.76%), and proline-alanine-serine-rich protein (PASrp) (52.80%), respectively, of Phaeoacremonium minimum tetramycovirus 1 (PmTmV1). MbPmV1 was grouped in a cluster with members of the genus Polymycovirus and was most closely related to PmTmV1 in the phylogenetic tree. Thus, we propose that MbPmV1 represents a novel species of the genus Polymycovirus, family Polymycoviridae. To our knowledge, this is the first report of a polymycovirus in a member of the genus Metarhizium.

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.

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.

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.

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.

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.

Class I myosin mediated endocytosis and polarization growth is essential for pathogenicity of Magnaporthe oryzae.

In eukaryotes, myosin provides the necessary impetus for a series of physiological processes, including organelle movement, cytoplasmic flow, cell division, and mitosis. Previously, three members of myosin were identified in Magnaporthe oryzae, with class II and class V myosins playing important roles in intracellular transport, fungal growth, and pathogenicity. However, limited is known about the biological function of the class I myosin protein in the rice blast fungus. Here, we found that Momyo1 is highly expressed during conidiation and infection. Functional characterization of this gene via RNA interference (RNAi) revealed that Momyo1 is required for vegetative growth, conidiation, melanin pigmentation, and pathogenicity of M. oryzae. The Momyo1 knockdown mutant is defective in formation of appressorium-like structures (ALS) at the hyphal tips. In addition, Momyo1 also displays defects on cell wall integrity, hyphal hydrophobicity, extracellular enzyme activities, endocytosis, and formation of the Spitzenkörper. Furthermore, Momyo1 was identified to physically interact with the MoShe4, a She4p/Dim1p orthologue potentially involved in endocytosis, polarization of the actin cytoskeleton. Overall, our findings provide a novel insight into the regulatory mechanism of Momyo1 that is involved in fungal growth, cell wall integrity, endocytosis, and virulence of M. oryzae. KEY POINTS: • Momyo1 is required for vegetative growth and pigmentation of M. oryzae. • Momyo1 is essential for cell wall integrity and endocytosis of M. oryzae. • Momyo1 is involved in hyphal surface hydrophobicity of M. oryzae.

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.

A cerato-platanin protein SsCP1 targets plant PR1 and contributes to virulence of Sclerotinia sclerotiorum.

Cerato-platanin proteins (CPs), which are secreted by filamentous fungi, are phytotoxic to host plants, but their functions have not been well defined to date. Here we characterized a CP (SsCP1) from the necrotrophic phytopathogen Sclerotinia sclerotiorum. Sscp1 transcripts accumulated during plant infection, and deletion of Sscp1 significantly reduced virulence. SsCP1 could induce significant cell death when expressed in Nicotiana benthamiana. Using yeast two-hybrid, GST pull-down, co-immunoprecipitation and bimolecular florescence complementation, we found that SsCP1 interacts with PR1 in the apoplast to facilitate infection by S. sclerotiorum. Overexpressing PR1 enhanced resistance to the wild-type strain, but not to the Sscp1 knockout strain of S. sclerotiorum. Sscp1-expressing transgenic plants showed increased concentrations of salicylic acid (SA) and higher levels of resistance to several plant pathogens (namely Botrytis cinerea, Alternaria brassicicola and Golovinomyces orontii). Our results suggest that SsCP1 is important for virulence of S. sclerotiorum and that it can be recognized by plants to trigger plant defense responses. Our results also suggest that the SA signaling pathway is involved in CP-mediated plant defense .

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.

Infection with a novel polymycovirus enhances growth, conidiation and sensitivity to UV-B irradiation of the entomopathogenic fungus Metarhizium anisopliae.

Metarhizium anisopliae is a well-studied entomopathogenic fungus that is widely used in biological control programs. The presence of polymycoviruses in this fungus is common, but their effects on fungal development and stress tolerance are not well understood. In this study, we report the discovery of a novel double-stranded RNA virus, named Metarhizium anisopliae polymycovirus 1 (MaPmV1), which comprises four dsRNAs ranging from 2.4 to 1.4 kbp in length. Phylogenetic analysis revealed that MaPmV1 belongs to the Polymycoviridae family. Biological comparison between MaPmV1-infected (Vi) and -free (Vf) isogenic lines showed that MaPmV1 remarkably enhances the growth rate and conidiation of the host fungus. The upregulation of growth- and conidiation-related genes in Vi strains supports this finding. In addition, MaPmV1 increases the sensitivity of the host to UV-B irradiation, which is evidenced by the downregulation of DNA damage repair genes in Vi strains. However, MaPmV1 does not appear to have any significant impact on the virulence of M. anisopliae. Furthermore, overexpression of individual viral proteins in M. anisopliae did not result in any significant phenotypic alterations, indicating that MaPmV1-mediated changes are not related to a single viral protein. Overall, our findings suggest that mycoviruses can be exploited to enhance fungal development in entomopathogenic fungi, which may lead to improved conidium production on a large scale.