Molecular characterization of a novel victorivirus (order Ghabrivirales, family Totiviridae) infecting Metarhizium anisopliae.

Here, we report the occurrence and complete genome sequence of a novel victorivirus infecting Metarhizium anisopliae, named "Metarhizium anisopliae victorivirus 1" (MaVV1). The genome is 5353 bp in length and contains two open reading frames (ORFs), encoding a coat protein and an RNA-dependent RNA polymerase (RdRp), that overlap at the octanucleotide sequence AUGAGUAA. These ORFs showed sequence similarity to the corresponding ORFs of Ustilaginoidea virens RNA virus L (68.23%) and Ustilaginoidea virens RNA virus 13 (58.11%), respectively, both of which belong to the family Totiviridae. Phylogenetic analysis based on RdRp sequences revealed that MaVV1 clustered with members of the genus Victorivirus. This is the first genome sequence reported for a virus belonging to the genus Victorivirus infecting the entomopathogenic fungus M. anisopliae.

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 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.

Expression of disease resistance in genetically modified grapevines correlates with the contents of viral sequences in the T-DNA and global genome methylation.

Increased tolerance to pathogens is an important goal in conventional and biotechnology-assisted grapevine breeding programs worldwide. Fungal and viral pathogens cause direct losses in berry production, but also affect the quality of the final products. Precision breeding strategies allow the introduction of resistance characters in elite cultivars, although the factors determining the plant's overall performance are not fully characterized. Grapevine plants expressing defense proteins, from fungal or plant origins, or of the coat protein gene of grapevine leafroll-associated virus 3 (GLRaV-3) were generated by Agrobacterium-mediated transformation of somatic embryos and shoot apical meristems. The responses of the transformed lines to pathogen challenges were investigated by biochemical, phytopathological and molecular methods. The expression of a Metarhizium anisopliae chitinase gene delayed pathogenesis and disease progression against the necrotrophic pathogen Botrytis cinerea. Modified lines expressing a Solanum nigrum osmotin-like protein also exhibited slower disease progression, but to a smaller extent. Grapevine lines carrying two hairpin-inducing constructs had lower GLRaV-3 titers when challenged by grafting, although disease symptoms and viral multiplication were detected. The levels of global genome methylation were determined for the genetically engineered lines, and correlation analyses demonstrated the association between higher levels of methylated DNA and larger portions of virus-derived sequences. Resistance expression was also negatively correlated with the contents of introduced viral sequences and genome methylation, indicating that the effectiveness of resistance strategies employing sequences of viral origin is subject to epigenetic regulation in grapevine.

Enzymatic activities and effects of mycovirus infection on the virulence of Metarhizium anisopliae in Rhipicephalus microplus.

The present study aimed to evaluate the pathogenic potential of different Metarhizium anisopliae s.l. isolates and to determine whether differences in enzymatic activities of proteases, lipases and chitinases and infection with mycoviruses affect the control of Rhipicephalus microplus achieved by these fungal isolates. Engorged female ticks were exposed to fungal suspensions. The lipolytic and proteolytic activities in the isolates were evaluated using chromogenic substrates and the chitinolytic activity was determined using fluorescent substrates. A gel zymography was performed to determine the approximate size of serine proteases released by M. anisopliae isolates. To detect mycoviral infections, dsRNA was digested using both RNAse A and S1 endonuclease; samples were analyzed on an agarose gel. Four of the five isolates tested were infected with mycovirus; however, the level of control of R. microplus ticks achieved with the only isolate free of infection (isolate CG 347) was low. This finding suggests that mycoviral infection does not affect the virulence of fungi against ticks. Although all five isolates were considered pathogenic to R. microplus, the best tick control and the highest levels of enzymatic activity were achieved with the isolates CG 629 and CG 148. The in vitro activities of lipases, proteases and chitinases produced by M. anisopliae s.l. differed among isolates and may be related to their virulence.