Field Trial Shows Virus-Based Biocontrol Tames Chestnut Blight Across Europe

Chestnut blight, one of the most devastating fungal diseases ever to strike forest trees, may have met its match in a virus that lives inside its own cells. A new field experiment spanning Switzerland, Croatia, and North Macedonia has provided the most rigorous real-world test yet of virus-mediated biological control against the chestnut blight fungus Cryphonectria parasitica, and the results are strikingly encouraging for European chestnut forests. The study, published in IMA Fungus, demonstrates that the mycovirus Cryphonectria hypovirus 1, known as CHV1, rapidly invades new fungal cankers under natural conditions, significantly slows their growth, and curtails the pathogen’s spore production, all of which improve the survival prospects of infected trees.

The chestnut blight fungus is an ascomycete that arrived in North America from Asia in the early twentieth century and drove the American chestnut, Castanea dentata, to functional extinction. When it reached Europe, first detected officially in 1938, the European chestnut suffered severe damage for roughly two decades before the epidemic unexpectedly mellowed. The reason was hypovirulence: infection of the fungus by CHV1, a parasitic RNA virus that saps the pathogen’s virulence. Fungal strains carrying the virus form superficial, callusing cankers that stop expanding, allowing the tree to survive. This natural system has since spread across many European chestnut-growing regions and stands as one of the few documented successes of biological control against an invasive forest pathogen.

Yet a critical question has lingered. CHV1 has no extracellular phase; it can move only within fungal hyphae, spreading vertically into asexual spores called conidia but never into sexual ascospores. Horizontal transmission between fungal strains requires hyphal fusion, or anastomosis, which is blocked by a vegetative incompatibility system. This system, governed by at least six unlinked diallelic vic loci, triggers contact-mediated cell death between hyphae that differ in their allele combinations. Laboratory experiments suggested these incompatibility barriers should severely restrict virus movement between fungal strains of different vegetative compatibility, or vc, types. Whether such barriers would cripple virus spread in real forests remained untested at scale.

To close this gap, researchers established a field experiment at six chestnut coppice sites with naturally established hypovirulence: Contone and Orselina in Switzerland, Kašt and Ozalj in Croatia, and Kalishte and Smolare in North Macedonia. In August 2014, they artificially initiated bark cankers on healthy chestnut sprouts using virus-free, genetically identifiable fungal isolates whose multilocus genotypes were absent from the local populations. The inoculated vc types ranged from rare to dominant in each resident population: EU-1 and EU-12 in Switzerland, EU-1 and EU-2 in Croatia, and only EU-12 in North Macedonia, where the entire local population belonged to that single type. Disease incidence at the sites ranged from 62 to 83 percent, and the pre-existing prevalence of CHV1 in cankers ranged from 25.8 percent at Smolare to 75 percent at Orselina, with all viral strains belonging to the Italian subtype.

Over 26 months, the team sampled the cankers at four time points, measuring canker dimensions, assessing activity and sporulation, and re-isolating the fungus from bark samples taken with a sterilized biopsy needle. Fungal isolates were characterized by vc type and ten microsatellite loci, while CHV1 infections were detected by the characteristic white culture morphology of virus-infected strains and confirmed by reverse transcription PCR and partial sequencing of the viral ORF-A region. Crucially, chi-square tests showed that the sampling procedure itself, which wounds the canker, did not influence the likelihood of virus infection, ruling out a potential artifact since C. parasitica is a wound parasite.

The speed of virus acquisition was remarkable. Just three months after inoculation, in November 2014, virus-infected fungal isolates were already recovered from the initially virus-free cankers at every site except Ozalj, with incidence ranging from 9.1 percent at Smolare to 58.3 percent at Kalishte. Infection rates climbed steadily thereafter. By October 2016, between 31.8 percent and 87.5 percent of cankers were virus-infected depending on the site, with Swiss sites reaching 64.1 and 69.2 percent and Croatian sites 74.2 and 86.1 percent. Sequence analysis revealed that in 66.2 percent of infected cankers, a single CHV1 strain had established and persisted over time, consistent with the view that canker infections are chronic events caused by a single transmission episode.

Most tellingly, the observed infection rates exceeded those predicted from laboratory-derived transmission rates across the vic loci. At Contone and Kašt, observed infections surpassed expectations from the very first sampling; at Orselina and Ozalj they caught up and overtook expectations by the end of the experiment. Spearman correlations between observed and expected rates weakened from weak to moderate at the early samplings to essentially negligible later on, indicating that the in vitro model lost predictive power as time passed. The researchers attribute the unexpectedly efficient virus spread to several factors: abundant hypovirulent inoculum in the resident populations, potentially fueled by virus-infected fungal growth on freshly dead chestnut wood; possible long-distance dispersal of conidia by rain splash, insects, birds, mites, or windborne dust; and a vegetative incompatibility system that appears far more permeable in nature than petri-dish experiments imply, particularly given the perennial nature of cankers that allow repeated contact opportunities over years.

The consequences for the trees were equally significant. In Switzerland, virus-infected cankers were significantly smaller than virus-free ones by October 2015, fourteen months after inoculation, and the gap widened a year later. In North Macedonia the effect appeared as early as May 2015 at Kalishte, though only at the final sampling at Smolare. Croatia showed no significant size difference by the experiment’s end, possibly because of late virus infections, lower local viral virulence, or more susceptible chestnut populations. Sporulation, however, was reduced at all sites, with mean stromata scores consistently higher on virus-free cankers. Passive cankers, those that had stopped expanding, were nearly five times more frequent among virus-infected cankers, 39.6 percent versus 8.8 percent, and the team observed callusing of hypovirus-infected cankers, signaling that host healing had begun. Given that virus-free cankers in the study expanded at an average of 16 centimeters per year, early virus infection appears essential for tree survival.

Microsatellite genotyping uncovered another key epidemiological insight: bark cankers are far more dynamic than they appear. By October 2016, the originally inoculated fungal genotype accounted for only 56 percent of isolates recovered, down from 67.2 percent a year earlier. Roughly 45 percent of cankers harbored mixed genotypes and 10 percent had been entirely colonized by new ones, most of which belonged to the same vc type as the original inoculant. Notably, this genotype turnover occurred at similar rates in virus-infected and virus-free cankers, meaning the virus itself did not block fungal immigration. Instead, the researchers argue, the constant influx of new fungal genotypes into existing cankers is an inherent feature of C. parasitica epidemiology and a major engine of CHV1 spread, since virus-carrying conidia germinate on colonized bark and fuse with the resident fungus, delivering the virus. Because the virus travels only with asexual spores and hyphae, this ongoing fungal traffic provides exactly the transmission route it needs.

The overall verdict is favorable for European chestnut blight management: CHV1 proved highly effective as a biological control agent, infecting new cankers of diverse vc types rapidly and suppressing their growth and sporulation. The authors caution, however, that their conclusions cannot be automatically extended to genetically distant vc types found outside Europe, such as those in Asia or the Caucasus, whose accidental introduction could still undermine the system. Maintaining phytosanitary measures to prevent new fungal genotypes from entering Europe remains essential to securing the long-term stability of this elegant three-way balance among tree, fungus, and virus.

Subject of Research: Virus-mediated biological control of the chestnut blight fungus Cryphonectria parasitica under field conditions

Article Title: Testing the efficiency of natural hypovirulence for biological control of chestnut blight under field conditions

Article References: Prospero, S., Schwarz, J. M., Ježić, M., Leigh, D. M., Ćurković-Perica, M., Idžojtić, M., Katanić, Z., Krstin, L., Nuskern, L., Pavičić, I., Poljak, I., Sotirovski, K., Risteski, M., Vuković, R., & Rigling, D. (2026). Testing the efficiency of natural hypovirulence for biological control of chestnut blight under field conditions. IMA Fungus, 17, Article e173675. https://doi.org/10.3897/imafungus.17.173675

Image Credits: AI Generated

DOI: 10.3897/imafungus.17.173675

Keywords: chestnut blight, Cryphonectria parasitica, CHV1, hypovirulence, biological control, mycovirus, vegetative incompatibility, Castanea sativa, forest pathology, field experiment, canker dynamics, Europe