Characterization of the Mycovirome of the Phytopathogenic Fungus, Neofusicoccum parvum

Abstract

Neofusicoccum parvum is a fungal plant-pathogen belonging to the family Botryosphaeriaceae, and is considered one of the most aggressive causal agents of the grapevine trunk disease (GTD) Botryosphaeria dieback. In this study, the mycovirome of a single strain of N. parvum (COLB) was characterized by high throughput sequencing analysis of total RNA and subsequent bioinformatic analyses. Contig annotations, genome completions, and phylogenetic analyses allowed us to describe six novel mycoviruses belonging to four different viral families. The virome is composed of two victoriviruses in the family Totiviridae, one alphaendornavirus in the family Endornaviridae, two mitoviruses in the family Mitoviridae, and one narnavirus belonging to the family Narnaviridae. The presence of the co-infecting viruses was confirmed by sequencing the RT-PCR products generated from total nucleic acids extracted from COLB. This study shows that the mycovirome of a single N. parvum strain is highly diverse and distinct from that previously described in N. parvum strains isolated from grapevines.

Keywords: Botryosphaeriaceae; Neofusicoccum parvum; fungi; grapevine trunk disease; high throughput sequencing; mycovirus.

Figure 1

Confirmation of the presence of the six mycoviruses in Neofusicoccum parvum COLB by specific RT-PCRs. Primer pairs used are specific for NpMV3 (lanes 1 and 2), NpMV2 (lanes 3 and 4), NpEV1 (lanes 5 and 6), NpVV2 (lanes 7 and 8), NpNV3 (lanes 9 and 10), and NpVV1 (11 and 12). Templates are total nucleic acids from COLB (lanes 1, 3, 5, 7, 9, and 11) and water (lanes 2, 4, 6, 8, 10, and 12). A molecular marker is in lane 13.

Figure 2

Genomic organization of mycoviruses identified in Neofusicoccum parvum isolates COLB. Ct 5, contig 5; NpEV1, neofusicoccum parvum endornavirus 1; Ct 14, contig 14; NpNV3, neofusicoccum parvum narnavirus 3; Ct 1, contig 1; NpMV2, neofusicoccum parvum mitovirus 2; Ct 2, contig 2; NpMV3, neofusicoccum parvum mitovirus 3; Ct 16, contig 16; NpVV1, neofusicoccum parvum victorivirus 1; Ct 9, contig 9; NpVV2, neofusicoccum parvum victorivirus 2. Numbers refer to genomic nucleotide positions. Open reading frames (ORF) are represented by boxes. Conserved RNA-dependent RNA polymerase (RdRp), RNA helicase (Hel), and glycosyl-transferase (GT) domains in the ORF1-encoded polyprotein are represented by striped shading, with the e-values associated with their identification using the conserved domain search program on the NCBI website. Missing sequences are represented by hyphenated lines (in non-coding regions) or dashed boxes (in ORFs).

Figure 3

Unrooted phylogenetic tree reconstructed from the alignment of the amino acid sequences of the RdRp domains of Endornaviridae representative members. The tree was constructed by the neighbor-joining method and the statistical significance of branches was evaluated by bootstrap analysis (1000 replicates). Only bootstrap values above 70% are indicated. The scale bar represents 5% amino acid divergence. Species belonging to the genera Alphaendornavirus and Betaendornavirus are identified respectively by a filled or an open circle. Neofusicoccum parvum endornavirus 1 is in bold and indicated by a black square.

Figure 4

Unrooted phylogenetic tree reconstructed from the alignment of the amino acid sequences of the RdRp of Narnaviridae and Mitoviridae representative members. The tree was constructed by the neighbor-joining method and the statistical significance of branches was evaluated by bootstrap analysis (1000 replicates). Only bootstrap values above 70% are indicated. The scale bar represents 20% amino acid divergence. Neofusicoccum parvum mitovirus 2 and 3 are in bold and indicated by black squares, and neofusicoccum parvum narnavirus 3 is in bold and indicated by a black triangle. The families to which belong the species are indicated in the right of the figure.

Figure 5

Unrooted phylogenetic tree reconstructed from the alignment of the amino acid sequences of the RdRp of Victorivirus representative members. The tree was constructed by the neighbor-joining method and the statistical significance of branches was evaluated by bootstrap analysis (1000 replicates). Only bootstrap values above 70% are indicated. The scale bar represents 5% amino acid divergence. Neofusicoccum parvum victorivirus 1 and 2 are in bold and indicated by black squares.