The Mycovirome in a Worldwide Collection of the Brown Rot Fungus Monilinia fructicola

Abstract

The fungus Monilinia fructicola is responsible for brown rot on stone and pome fruit and causes heavy yield losses both pre- and post-harvest. Several mycoviruses are known to infect fungal plant pathogens. In this study, a metagenomic approach was applied to obtain a comprehensive characterization of the mycovirome in a worldwide collection of 58 M. fructicola strains. Deep sequencing of double-stranded (ds)RNA extracts revealed a great abundance and variety of mycoviruses. A total of 32 phylogenetically distinct positive-sense (+) single-stranded (ss)RNA viruses were identified. They included twelve mitoviruses, one in the proposed family Splipalmiviridae, and twelve botourmiaviruses (phylum Lenarviricota), eleven of which were novel viral species; two hypoviruses, three in the proposed family Fusariviridae, and one barnavirus (phylum Pisuviricota); as well as one novel beny-like virus (phylum Kitrinoviricota), the first one identified in Ascomycetes. A partial sequence of a new putative ssDNA mycovirus related to viruses within the Parvoviridae family was detected in a M. fructicola isolate from Serbia. The availability of genomic sequences of mycoviruses will serve as a solid basis for further research aimed at deepening the knowledge on virus-host and virus-virus interactions and to explore their potential as biocontrol agents against brown rot disease.

Keywords: +ssRNA virus; barnavirus; benyvirus; botourmiavirus; fusarivirus; hypovirus; mitovirus; mycovirus; parvovirus; splipalmivirus; stone fruit.

Figure 1

Maximum likelihood phylogenetic tree based on multiple amino acid sequence alignment of the RdRp proteins of viruses in the family Mitoviridae (cyan) and the proposed family Splipalmiviridae (pink; [14]), and their relationships with the closest families Leviviridae, Narnaviridae, and Botourmiaviridae in the phylum Lenarviricota. Mycoviruses found in this work are in bold red. The numbers on nodes, shown for values greater than 50%, are the results of 1000 bootstrap analyses. The scale bar represents a genetic distance of 0.5 amino acid substitutions per site. The list of accession numbers of the analysed sequences is in Table S1.

Figure 2

Maximum likelihood phylogenetic tree based on multiple amino acid sequence alignment of the RdRp proteins of viruses in the family Botourmiaviridae and its relationships with the closest families Narnaviridae, Leviviridae, Splipalmiviridae, and Mitoviridae in the phylum Lenarviricota. The genera in the Botourmiaviridae family are represented with different coloured boxes: Ourmiavirus (OV, yellow), Rhizoulivirus (RV, red), Magoulivirus (purple), Scleroulivirus (cyan), Botoulivirus (green), and Penoulivirus (pink). The type species is in bold black. Mycoviruses found in this work are in bold red. Single or double asterisks indicate new proposed species and genera, respectively. The numbers on nodes, shown for values greater than 50%, are the results of 1000 bootstrap analyses. The scale bar represents a genetic distance of 0.5 amino acid substitutions per site. The list of accession numbers of the analysed sequences is in the Table S1.

Figure 3

Maximum likelihood phylogenetic tree based on multiple amino acid sequence alignment of the RdRp proteins of viruses in the family Hypoviridae and its relationship with the closely related proposed family Fusariviridae. The three genera in the Hypoviridae family are represented with different coloured boxes: Alphahypovirus (green), Betahypovirus (red), and the proposed genus Gammahypovirus (cyan). The Beihai hypo-like virus and Beihei sipunculid worm virus 6 (yellow) are proposed as members of an additional genus [65]. The type species are in bold black. Mycoviruses found in this work are in bold red. The numbers on nodes, shown for values greater than 50%, are the results of 1000 bootstrap analyses. The scale bar represents a genetic distance of 0.2 amino acid substitutions per site. The list of accession numbers of the analysed sequences is in Table S1.

Figure 4

Maximum likelihood phylogenetic tree based on multiple amino acid sequence alignment of the RdRp proteins of viruses in the proposed Fusariviridae family and its relationship with the closely related family Hypoviridae. Coloured boxes indicate Group 1 (yellow) and Group 2 (purple) of the fusariviruses [44]. Mycoviruses found in this work are in bold red. The numbers on nodes, shown for values greater than 50%, are the results of 1000 bootstrap analyses. The scale bar represents a genetic distance of 0.2 amino acid substitutions per site. The list of accession numbers of the analysed sequences is in Table S1.

Figure 5

Maximum likelihood phylogenetic tree based on multiple amino acid sequence alignment of the replicase protein of Monilinia fructicola barnavirus 1 and viruses of the genus Barnavirus (Barnaviridae family) reported in Basidiomycetes (B; yellow) and Ascomycetes (A; green), and the closely related members of the Solemoviridae family (grey). The type species is in bold black. The mycovirus found in this work is in bold red. The number on nodes, shown for values greater than 50%, are the results of 1000 bootstrap analyses. The scale bar represents a genetic distance of 0.2 amino acid substitutions per site. The list of accession numbers of the analysed sequences is in Table S1.

Figure 6

Maximum likelihood phylogenetic tree based on multiple amino acid sequence alignment of the nonstructural replication-associated protein of Monilinia fructicola beny-like virus 1 and beny-like viruses (Benyviridae family) from various hosts, including fungi (blue), insects (pink), and plants (green). The Hepeviridae family (grey) was used as outgroup. The type species is in bold black. The mycovirus found in this work is in bold red. The asterisk indicates the newly proposed species. The number on the nodes, shown for values greater than 50%, are the results of 1000 bootstrap analyses. The scale bar represents a genetic distance of 0.2 amino acid substitutions per site. The list of accession numbers of the analysed sequences is in Table S1.

Figure 7

Detection rate of viral families and single viruses in 12 pools of Monilinia fructicola isolates in RPKM (reads normalized per kilobases of viral genome length and millions of total reads mapped on the virome). (A) Mitoviriridae; (B) Splipalmiviridae; (C) Botourmiaviridae; (D) Hypoviridae; (E) Fusariviridae; (F) Benyviridae; (G) Barnaviridae; (H) Parvoviridae.