Canker Development and Biocontrol Potential of CHV-1 Infected English Isolates of Cryphonectria parasitica Is Dependent on the Virus Concentration and the Compatibility of the Fungal Inoculums

Abstract

Biological control of Cryphonectria parasitica fungus, causal agent of chestnut blight, by virus infection (hypovirulence) has been shown to be an effective control strategy against chestnut blight in Europe and some parts of North America. The most studied mycovirus is the Cryphonectria hypovirus 1 (CHV-1) type species of the Hypoviridae family. To efficiently provide biocontrol, the virus must be able to induce hypovirulence in its fungal host in chestnut trees. Here, two different CHV-1 subtype I virus strains (E-5 and L-18), gained by transmissions, were tested for their hypovirulence induction, biocontrol potential, and transmission between vegetatively compatible (VCG) and incompatible fungal isolate groups in sweet chestnut seedlings and branches. Both strains of CHV-1 showed great biocontrol potential and could protect trees by efficiently transmitting CHV-1 by hyphal anastomosis between fungal isolates of the same VCG and converting virulent to hypovirulent cankers. The hypovirulent effect was positively correlated with the virus concentration, tested by four different reverse-transcription PCRs, two end-point and two real-time methods, one of which represents a newly developed real-time PCR for the detection and quantification of CHV-1.

Keywords: Cryphonectria hypovirus 1; England; branches; compatibility; concentration; preservations; real-time PCR; seedlings; transmissions.

Figure 1

Regression equation relating the CHV-1 virus copy number with threshold cycle values.

Figure 2

Lesion area produced by virus-infected and virus-free C. parasitica isolates. Points with error bars represent estimated marginal means with 95% confidence intervals. Lettering indicates significant differences by treatment. (A) Seedlings. (B) Branch segments. See Material and Methods third paragraph for experimental details.

Figure 3

Lesion area 53 days after individual inoculation (assay I) with ten isolates of Cryphonectria parasitica (1–3 infected with the hypovirus CHV1-M2273, 4–6 infected with CHV1-M2357, and 8–11 virus-free isolates) and a PDA control (number 7). (Eleven treatments). Bars with the same letter are not significantly different based on Tukey′s test. (A) Seedlings (n = 44). (B) Branch segments (n = 33). See Material and Methods third paragraph for experimental details.

Figure 4

Lesion area by primary inoculation and challenge inoculation with virus or without virus (control) using seedlings. Points with error bars represent estimated marginal means with 95% confidence intervals. Lettering indicates significant differences between challenge inoculation with virus compared to the control. See Material and Methods third paragraph for experimental details.

Figure 5

Virus concentration within the re-isolations after 53 days post individual inoculation (assay I, (A–D) using seedings, (E–H) using branch segments) with ten isolates of Cryphonectria parasitica (1–3 infected with the hypovirus CHV1-M2273, 4–6 infected with CHV1-M2357, and 8–11 virus-free isolates) and a PDA control (number 7). (Eleven treatments). Bars with the same letter are not significantly different based on a Tukey′s test. (A) Seedlings (n = 44). (B) Branch segments (n = 33). Bars with an asterisk indicate significant differences against the same isolate treatment analysed using the Qiagen Extract End-point PCR method for the CHV-1 mycovirus.