Identification of a novel hypovirulence-inducing ourmia-like mycovirus from Fusarium solani causing ginseng (Panax ginseng) root rot

Abstract

Introduction: Fusarium solani is a widespread plant pathogen known to damage numerous crops, including causing severe root rot in Panax ginseng. In this study, we identified a novel ourmia-like mycovirus in F. solani, named "Fusarium solani ourmia-like virus 1" (FsoOLV1). We demonstrated that FsoOLV1 confers hypovirulence in its host F. solani and three other ginseng root rot pathogens, F. oxysporum, F. proliferatum, and F. verticillioides. Additionally, we verified its horizontal and vertical transmission capabilities.

Methods: FsoOLV1 was discovered in F. solani strain SJH 2-4 using high-throughput sequencing. The genome sequence of FsoOLV1 was obtained through RT-PCR and RACE. Virus elimination was conducted to assess the effect of FsoOLV1 on fungal virulence. Protoplast transfection experiments were performed to evaluate the impact of the virus on other ginseng root rot pathogens. Horizontal and vertical transmission studies were also carried out to examine the spread of the virus.

Results: The genome of FsoOLV1 is 2,801 nucleotides (nt) in length with a GC content of 47.05%. It encodes a 750 amino acid RNA-dependent RNA polymerase (RdRp), with a molecular weight of approximately 84.84 kDa. Phylogenetic analysis indicated that FsoOLV1 clusters within a clade containing the Magoulivirus genus in the Botourmiaviridae family. Curing FsoOLV1 from the fungal host strain revealed that the mycovirus plays a role in reducing the virulence of the F. solani strain SJH 2-4. Furthermore, protoplast transfection revealed that FsoOLV1 can significantly reduce the virulence of other ginseng root rot pathogens, including F. oxysporum, F. proliferatum, and F. verticillioides. Additionally, FsoOLV1 is capable of horizontal transmission between F. solani strains and vertical transmission to the next generation via conidia.

Discussion: This study presents the first hypovirulent ourmia-like virus, FsoOLV1, which reduces the virulence of F. solani and other ginseng root rot pathogens. Our findings suggest that FsoOLV1 could serve as a promising biological agent for controlling ginseng root rot. This research not only expands the diversity of known hypovirulent mycoviruses but also provides a potential candidate for controlling Fusarium diseases in ginseng cultivation.

Keywords: Fusarium solani; ginseng root rot; hypovirulence; mycovirus; ourmia-like virus.

FIGURE 1

Biological characteristics and dsRNA profile of Fusarium solani strain SJH 2-4. (A) Colony morphology of F. solani strain SJH 2-4 compared with the virulent strain TH 7-2 (cultured on PDA for 5 days at 28°C). (B) Growth rate comparison between strains SJH 2-4 and TH 7-2. (C) Pathogenicity of strains SJH 2-4 and TH 7-2 on ginseng roots (7 days post-inoculation). The mock group served as a blank control. (D) Lesion diameters caused by strains SJH 2-4 and TH 7-2. Error bars represent the standard deviation (SD) of the mean. Different lowercase letters above the columns indicate significant differences (p < 0.05). (E) Agarose gel electrophoresis of the dsRNA extracted from strain SJH 2-4, treated with DNase I and S1 nuclease. M, molecular weight marker.

FIGURE 2

Genomic features of Fusarium solani ourmia-like virus 1 (FsoOLV1). (A) Schematic representation of the genomic organization of FsoOLV1. The open reading frame (ORF) encoding the RNA-dependent RNA polymerase (RdRp) domain is depicted as a pink box (55–2,310 nt). (B) Potential secondary structures of the 5’- (left) and 3’- (right) termini of FsoOLV1. (C) Multiple sequence alignment of the amino acid sequences of RdRp proteins encoded by FsoOLV1 and other selected ourmia-like viruses. Seven conserved RdRp motifs are indicated with Roman numerals I to VII. Abbreviations of the viruses are as follows: FsoOLV1, Fusarium solani ourmia-like virus 1; PvlaOLV 13, Plasmopara viticola lesion associated ourmia-like virus 13; PvlaOLV 19, Plasmopara viticola lesion associated ourmia-like virus 19; PBV1, Pestalotiopsis botourmiavirus 1; CgOLV 1, Colletotrichum gloeosporioides ourmia-like virus 1.

FIGURE 3

Phylogenetic analysis of FsoOLV1 and other related viruses based on RdRp sequences. Phylogenetic tree constructed using the maximum likelihood method with 1,000 bootstrap replicates. The virus studied in this paper is marked with a red dot. The scale bar represents a genetic distance of 1.

FIGURE 4

Effect of FsoOLV1 on the biological characteristics of Fusarium solani strain SJH 2-4. Colony morphology (A), hyphal tip morphology (B), detailed view of hyphal tips (C), and macrocondium morphology (D) of the original strain SJH 2-4 and three virus-free strains (2-4 VF1-3). Scale bars = 20 μm. Comparison of the mycelial growth rate (E), spore yield (F), spore germination rate (G), and mycelial biomass (H) of SJH 2-4 and the virus-free strains. Error bars represent the standard deviation (SD) of the means. Different lowercase letters above columns indicate significant differences (p < 0.05).

FIGURE 5

Pathogenicity detection of Fusarium solani strain SJH 2-4 and virus-free strains (2-4 VF1-3). (A) Symptoms observed on ginseng roots inoculated with the original strain SJH 2-4 and three virus-free strains. The mock group served as a blank control. (B) Lesion diameters induced on ginseng roots. Error bars represent the standard deviation (SD) of the mean. Different lowercase letters above columns indicate significant differences (p < 0.05).

FIGURE 6

Horizontal and vertical transmission of FsoOLV1. (A,B) Horizontal transmission of FsoOLV1 from the donor strain SJH 2-4 to the recipient strains 2-4 VF (A) and TH7-2 (B) via hyphal anastomosis. Stars represent the regions where mycelial plugs were taken and cultured for subsequent RT-PCR detection. (C) RT-PCR detection of FsoOLV1 in the recipient strains using virus-specific primers. (D) Vertical transmission of FsoOLV1. Single conidial sub-isolates from the original strain SJH 2-4 were tested for FsoOLV1 presence using RT-PCR.

FIGURE 7

Effects of FsoOLV1 on fungal morphology in four Fusarium species. (A) RT-PCR detection of FsoOLV1 in four Fusarium species using virus-specific primers. M, 2,000 bp DNA marker. (B) Colony morphology of the original recipient strains and FsoOLV1-infected (VI) strains after 7 days of culture on PDA at 28°C.

FIGURE 8

FsoOLV1 induces hypovirulent phenotypes in four Fusarium species. (A) Virulence assay of four original Fusarium species and FsoOLV1-infected (VI) strains on ginseng roots. (B) Lesion diameters induced on ginseng roots. Error bars represent the standard deviation (SD) of the means. Different lowercase letters above columns indicate significant differences (p < 0.05).