Transmission of Cryphonectria Hypovirus 1 (CHV1) to Cryphonectria radicalis and In Vitro and In Vivo Testing of Its Potential for Use as Biocontrol Against C. parasitica

Abstract

Cryphonectria hypovirus 1 (CHV1) is successful in controlling Cryphonectria parasitica, the causal agent of chestnut blight, but little is known regarding its transmission to other fungi, for example the European Cryphonectria radicalis. In this study, CHV1 was transmitted (circa 200,000-800,000 copies/microliter) to seven C. radicalis isolates from infected C. parasitica. Reverse transmission to virus-free C. parasitica (European 74 testers collection) was achieved, although it was less successful (250-55,000 copies/µL) and was dependent on the vegetative compatibility (VC) group. In C. radicalis, the virus infection led to colony colour change from pink to white and smaller colonies, dependent on the virus concentration. The virus was concentrated in the colony edges, and vertically transmitted to 77% of conidia. However, several in vitro experiments demonstrated that C. radicalis was always outcompeted by the blight fungus, only suppressing the pathogen between its 25-50% inoculum level. It presented good secondary capture only when acting as a pioneer. Two types of in planta assays (individual and challenge inoculations) were undertaken. Cryphonectria radicalis behaved as a saprotroph, while chestnut blight fungus behaved as an aggressive pathogen, and lesions after treatment with C. radicalis were no smaller in general, only when using cut branches. Overall, the results showed that infected C. radicalis was unable to control cankers.

Keywords: Cryphonectria parasitica; Cryphonectria radicalis; biocontrol; hypovirus 1 (CHV1); transmissions; virulence.

Figure 1

Pictures of 7 days old cultures of the seven CHV1 mycovirus infected C. radicalis isolates (subbed from the centre or the edge) and the respective non-infected isogenic cultures, with indication of the copy number of the virus.

Figure 2

Differential competition of chestnut blight fungus, Cryphonectria parasitica EU9 vegetative compatibility group, interactions with Cryphonectria radicalis isolates LES362 and BOS158, non-infected or infected (highlighted in grey) by the CHV1 mycovirus. The significance of the decrease of C. parasitica growth areas as its proportion is lower and indicated by the p-value. Relative Crowding Coefficient, when over one, C. parasitica is outcompeting C. radicalis. See Section 4.4 for experimental details.

Figure 3

Primary resource capture capabilities of chestnut blight fungus, Cryphonectria parasitica EU9 vegetative compatibility group (A,C,E,G), interaction with Cryphonectria radicalis isolates LES362 and BOS158, non-infected or infected (highlighted in grey) by the CHV1 mycovirus (B,D,F,H). The significance of the decrease of each fungus growth areas in pairwise confrontation with the other fungus is indicated by the p-value of a paired-samples t-test. See Section 4.5 for experimental details. Continuous and dashed lanes respectively indicate the area colonised of each species growing in isolation or confronted with the other species.

Figure 4

Secondary resource capture capabilities of chestnut blight fungus, Cryphonectria parasitica EU9 vegetative compatibility group (A,F), interaction with Cryphonectria radicalis isolates LES362 and BOS158, non-infected or infected (highlighted in grey) by the CHV1 mycovirus (B–E,G–J), when acting as a pioneer or a competitor. See Section 4.6 for experimental details.

Figure 5

Lesion area (mean ± SE) produced by virus-infected and virus-free C. parasitica and C. radicalis or PDA control. Lettering indicates significant differences by treatment. (A) Branch segments; (B) saplings. See Section 4 last epigraph for experimental details.

Figure 6

Lesion area (mean ± SE) produced by virus-infected (white bars) and virus-free (black bars) C. parasitica and C. radicalis or PDA control (white bar). Lettering indicates significant differences by treatment. (A) Branch segments; (B) saplings. See Section 4 last epigraph for experimental details.

Figure 7

Lesion area (mean ± SE) by primary inoculation and challenge inoculation (assay II) with virus or without virus (control) using branches (A,B) or saplings (C,D). Different lettering indicates significant differences between the different challenge inoculations results. See Section 4 last epigraph for experimental details.

Figure 8

Lesion area (mean ± SE) by primary inoculation and challenge inoculation (assay II repeated, targeted) with virus or without virus (control) using branches (A,B) or saplings (C,D). Different lettering indicates significant differences between the different challenge inoculations results. See Section 4 last epigraph for experimental details.