Studies on the Virome of the Entomopathogenic Fungus Beauveria bassiana Reveal Novel dsRNA Elements and Mild Hypervirulence

Abstract

The entomopathogenic fungus Beauveria bassiana has a wide host range and is used as a biocontrol agent against arthropod pests. Mycoviruses have been described in phytopathogenic fungi while in entomopathogenic fungi their presence has been reported only rarely. Here we show that 21.3% of a collection of B. bassiana isolates sourced from worldwide locations, harbor dsRNA elements. Molecular characterization of these elements revealed the prevalence of mycoviruses belonging to the Partitiviridae and Totiviridae families, the smallest reported virus to date, belonging to the family Narnaviridae, and viruses unassigned to a family or genus. Of particular importance is the discovery of members of a newly proposed family Polymycoviridae in B. bassiana. Polymycoviruses, previously designated as tetramycoviruses, consist of four non-conventionally encapsidated capped dsRNAs. The presence of additional non-homologous genomic segments in B. bassiana polymycoviruses and other fungi illustrates the unprecedented dynamic nature of the viral genome. Finally, a comparison of virus-free and virus-infected isogenic lines derived from an exemplar B. bassiana isolate revealed a mild hypervirulent effect of mycoviruses on the growth of their host isolate and on its pathogenicity against the greater wax moth Galleria mellonella, highlighting for the first time the potential of mycoviruses as enhancers of biocontrol agents.

Fig 1. Polymycoviruses in Beauveria bassiana.

(a) 1.5% (^w/_v) agarose gel electrophoresis of dsRNA extracted from B. bassiana isolates EABb 92/11-Dm, IMI 391043 and ATHUM 4946 harboring BbPmV-1, BbPmV-2 and BbPmV-3 respectively. Lane 1 contains the DNA marker Hyperladder I (Bioline), the sizes of which are shown to the left of the gel. (b) Schematic representation of the genomic organization of BbPmV-1. The BbPmV-1 genome consist of four dsRNAs, each containing one ORF (grey boxes) flanked by 5’- and 3’-UTRs (black boxes). (c) Schematic representation of the genomic organization of the proposed family Polymycoviridae illustrating known or predicted features of the proteins encoded by the four major dsRNAs. Additional sequenced elements, unique for selected polymycoviruses, each containing one ORF (grey boxes) flanked by 5’- and 3’-UTRs (black boxes) are shown and include BdPmV-1 dsRNA 5, CcPmV-1 dsRNA 5, BbPmV-2 dsRNAs 6 and 7, and AltPmV-1 dsRNAs 5 and 6.

Fig 2. A small Narna-like virus in Beauveria bassiana.

(a) 1% (^w/_v) agarose gel electrophoresis and northern blot hybridization of dsRNA extracted from B. bassiana isolate SP R 159, using virus purification (lane 2) and LiCl dsRNA extraction (lane 4). Lane 6 contains partitivirus BbPV-1, as a negative control. Hybridization was carried out using specific probes for BbSNLV-SR-1: BbSNLV and SR 1 and 2 are indicated by arrows. (b) Schematic representation of the genomic organization of BbSNLV. The BbSNLV genome consists of a single RNA containing one ORF (grey box) flanked by 5’- and 3’-UTRs (black boxes). (c) Predicted secondary structure of the BbSNLV 5’- and 3’-UTRs and the (+) strand of BbSNLV-SR-1 using mfold (folding temperature 25°C). (d) Relative RNA levels of the positive and negative strands BbSNLV-SRs within total fungal ssRNA extracts, as shown by RT-qPCR amplification. At least three independent repetitions were performed in triplicate and error bars represent standard deviation. (e) A comparative alignment of the 5’- and 3’-terminal sequences of BbSNLV and BbSNLV-SR-1. Asterisks signify identical nucleotides. (f) A comparison of the conserved motifs of the RdRP in BbSNLV and other narnaviruses (S2 Table). Numbers within the brackets indicate the number of aa not shown. Asterisks signify identical aa residues, colons signify highly conserved residues and single dots signify less conserved but related residues. (g) Maximum likelihood phylogenetic tree created based on the alignment of RdRP sequences of narnaviruses (S2 Table) using the LG+G+I+F substitution model. Branches with bootstrap support lower that 50% were collapsed. At the end of the branches, red, orange, green and purple indicate that the virus infects filamentous fungi, yeast, oomycetes and kinetoplastids respectively. BbSNLV is indicated by an arrow.

Fig 3. Comparison of the Beauveria bassiana EABb 92/11-DmT and EABb 92/11-DmC; virus-transfected and virus-free isogenic lines.

Comparison of the fungal growth in solid and liquid medium; (a) radial growth and (b) biomass production of EABb 92/11-DmC and EABb 92/11-DmT. At least three independent repetitions were performed in triplicate (radial growth) or duplicate (biomass production) and error bars represent standard deviation. A yellow background indicates the time points when the difference in growth is statistically significant (Student’s t test, P-value < 0.05). (c) Mean survival curves of G. mellonella larvae infected with the EABb 92/11-DmC and EABb 92/11-DmT isogenic lines. At least three independent repetitions were performed in triplicate. (d) Colonies of the EABb 92/11-Dm, EABb 92/11-DmC and EABb 92/11-DmT grown on Czapek-Dox CM.