Molecular and biological characterization of a partitivirus from Paecilomyces variotii

Abstract

Paeciliomyces variotii is a thermo-tolerant, ubiquitous fungus commonly found in food products, indoor environments, soil and clinical samples. It is a well-known biocontrol agent used against phytopathogenic fungi and its metabolites have many industrial applications. Rare reports of P. variotii-related human infections have been found in the medical literature. In this study, we report for the first time the infection of P. variotii isolated from a soil sample collected in a rice field with a double-stranded RNA virus, Paeciliomyces variotii partitivirus 1 (PvPV-1) in the family Partitiviridae. P. variotii harboured icosahedral virus particles 30 nm in diameter with two dsRNA segments 1758 and 1356 bp long. Both dsRNA1 and dsRNA2 have a single open reading frame encoding proteins of 63 and 40 kDa, respectively. These proteins have significant similarity to the RNA-dependent RNA polymerase and capsid protein encoded by the genomic segments of several viruses from the family Partitiviridae. Phylogenetic analysis revealed that PvPV-1 belongs to the family Partitiviridae but in an unclassified group/genus, tentatively nominated Zetapartitivirus. PvPV-1 was found to increase the growth rate of the host fungus, as indicated by time course experiments performed on a range of different media for virus-infected and virus-free isogenic lines. Further, dual-culture assays performed for both isogenic lines confirmed the antagonistic potential of P. variotii against other phytopathogenic fungi. The findings of this study assist us in understanding P. variotii as a potential biocontrol agent, together with plant-fungus-virus interactions.

Keywords: Paecilomyces variotii; Partitiviridae; biological control; double-stranded RNA; growth; metabolism; zetapartitivirus.

Fig. 1.

PvPV-1 components. (a) Colony morphology of P. variotii isolate 15R on PDA. (b) TEM visualization of icosahedral VLPs 30 nm in diameter extracted from P. variotii. (c) Electrophoretic profile of the dsRNA elements extracted from P. variotii on a 1 % (w/v) agarose gel, showing two dsRNA bands approximately 1.5 and 1.9 kbp in size. The molecular sizes of λ-EcoT141-digested DNA markers are indicated on the left of the gel. (d) Electrophoretic profile of the VLPs on 10 % (w/v) SDS-PAGE analysis of the purified virus particles showing a CP approximately 40 kDa in mass. The molecular sizes of PAGE Ruler unstained protein ladders are indicated on the left of the gel.

Fig. 2.

PvPV-1 genomic organization. (a) Schematic representation of PvPV-1 dsRNA 1 and dsRNA 2. Each dsRNA is shown as a black line and each ORF as a grey box. (b) Nucleotide sequence alignment of the 5′- and 3′-termini of the coding strands of the two PvPV-1 dsRNAs using MAFFT. Asterisks signify identical nucleotides. (c) Amino acid sequence alignment of the core RdRP motifs (III–VIII) of PvPV-1 and selected members in the family Partitiviridae using MAFFT. Asterisks signify identical residues; colons signify highly conserved residues and single dots signify less conserved but related residues.

Fig. 3.

PvPV-1 phylogeny. Phylogenetic analysis of PvPV-1 and selected members of the family Partitiviridae based on their RdRP amino acid sequences. A multiple alignment of RdRP amino acid sequences was produced using muscle as implemented using mega 11. A neighbour-joining (NJ) phylogenetic tree was constructed using MEGA 11. Bootstrap percentages (1000 replicates) over 50 % are shown. Tips labelled with brown, green and blue shapes indicate that the virus host is respectively fungal, plant or protozoon. The orange rhombus indicates the position of PvPV-1.

Fig. 4.

PvPV-1 elimination. (a) Agarose gel electrophoresis of the PvPV-1 RdRP amplicon following RT-PCR of P. variotii virus-infected (VI) and virus-free (VF) isogenic lines before and after digestion with EcoRV. The molecular sizes of the Thermo Fisher Scientific 1 kbp DNA marker are indicated on the left of the gels. (b) The PvPV-1 RdRP amplicon sequence (466 bp) showing the forward and reverse primers (highlighted in yellow) and the EcoRV restriction enzyme site (highlighted in magenta). (c) Colony morphologies of P. variotii isogenic lines on PDA.

Fig. 5.

Radial growth of P. variotii isogenic lines. (a) Colony morphologies and (b) radial growth of P. variotii virus-infected (VI) and virus-free (VF) isogenic lines on different media, including PDA, SDA, YES, CDA-CM and CDA-MM, over a period of 5 days. Asterisks indicate statistical significance following two-way ANOVA: *, P-value<0.05; **, P-value <0.01; ***, P-value<0.001.

Fig. 6.

Biomass and metabolism of P. variotii isogenic lines. (a) Biomass production of P. variotii virus-infected (VI), virus-free (VF) and virus recipient (R5) isogenic lines in PBD. Asterisks indicate statistical significance following Student’s t-test: **, P-value <0.01; ***, P-value <0.001. (b) Metabolic activity of P. variotii isogenic lines in MEB and YES. (c) Metabolic activity of P. variotii isogenic lines in CD-MM with and without carbon and nitrogen sources. Asterisks indicate statistical significance following two-way ANOVA: **, P-value <0.01.

Fig. 7.

Antagonistic potential of P. variotii isogenic lines. (a) Dual-culture assay and (b) percentage inhibition of P. variotii virus-infected (VI) and virus-free (VF) isogenic lines against three plant pathogenic fungi, A. terreus, Hypoxylon spp. and P. citrinum. The blue and red arrows in (a) indicate the colony diameter of the fungal pathogens in control (A) and treated (B) groups, respectively, as measured during exemplar dual-culture assays to calculate percentage inhibition in (b). Two-way ANOVA indicated an overall statistical significance with P-value <0.01 when comparing P. variotii isogenic lines.