Since its introduction into Europe in the first half of the 20th century, Cryphonectria parasitica has been gradually spreading across the natural range of the sweet chestnut (Castanea sativa Mill.), infecting the trees and causing lethal bark cankers. Serendipitously, a hyperparasitic Cryphonectria hypovirus 1 (CHV1), which attenuates C. parasitica virulence in combination with more tolerant European chestnut species, was able to ward off the worst effect of the disease. In North America, unfortunately, the native Castanea dentata is now functionally extinct since it occurs only as root sprouts in eastern deciduous forests where it was once dominant. In our work, we investigated changes in C. parasitica populations over time and the regional variability in chestnut populations’ tolerance toward the blight disease. While vegetative compatibility (vc) type diversity and prevalence of hypovirulence remained similar as in previous studies, in the Buje population, unlike in previous studies, we were unable to find any hypovirulent fungal strains. The most common vegetative compatibility types (vc types) were EU-1, EU-2 and EU-12. However, several rare EU-types were found, including one previously unreported: EU-46. By inoculating several C. parasitica strains on tree stems from several chestnut populations, we observed that the induced lesion size was affected by the type of inoculum (CHV1-free or CHV1-infected), genotype-related individual chestnut stem and chestnut stem population of origin-related variability. The largest lesions were induced by CHV1-free fungal isolate DOB-G: 20.13 cm² (95% C.I. 18.10–22.15) and the smallest by CHV1-infected L14/EP713: 2.49 cm² (95% C.I. 1.59–3.39). Surprisingly, the size of the lesions induced by other CHV1-infected strains fell somewhere in between these extremes. The size of induced lesions was dependent on the population of origin as well and ranged from 11.60 cm² (95% C.I. 9.87–13.33) for stems from the Moslavačka gora population to 17.75 cm² (95% C.I. 15.63–19.87) for stems from Ozalj.

Marginal populations are usually smaller and more isolated and grow in less favourable conditions than those at the distribution centre. The variability of these populations is of high importance, as it can support the adaptations needed for the conditions that they grow in. In this research, the morphological variability of eight Tatar maple (Acer tataricum L. subsp. tataricum) populations was analysed. Tatar maple is an insect-pollinated and wind-dispersed shrub/tree, whose northwestern distribution edge is in southeastern Europe. Morphometric methods were used to analyse the variability of the populations using leaf and fruit morphology. The research revealed significant differences between and within populations. Furthermore, differences in the distribution of the total variability were noted, which suggest that different evolutionarily factors affect different plant traits. Correlation analysis confirmed a weak dependency between the vegetative and generative traits. In addition, no evidence was found for the presence of isolation by environment (IBE). However, the Mantel test for isolation by distance (IBD) was significant for the leaf morphometric traits and non-significant for the fruit morphometric traits. Being the marginal leading-edge populations, they are younger and were less likely to have had time for adaptation to local environments, which would have resulted in the development of IBE. Overall, edge populations of Tatar maple were characterised by great morphological variability, which helps these populations in their response to the intensive selective pressures they face in their environment.

Dutch elm disease (DED) was originally caused by the ascomycete Ophiostoma ulmi, which has been replaced by a more virulent species, O. novo-ulmi, divided into subsp. novo-ulmi and subsp. americana. Permeable reproductive barriers, a period of co-occurrence of O. ulmi and O. novo-ulmi, and the current overlap of O. novo-ulmi subspecies have been important in shaping the present O. novo-ulmi populations in Europe, which were initially clonal, predominantly of the MAT-2 type. This study confirmed the persistence of O. novo-ulmi in Croatia over the years, although at some forest sites, the diseased elms were not detected. The methodology used to assess changes in O. novo-ulmi populations was based on the col1 and cu genes, which have subspecies-specific nucleotide differences, analysis of MAT idiomorphs, and temperature-growth responses. The col1 and cu gene sequencing did not reveal a change in the number of isolates with the recombinant col1/cu genotype over 10 years (2012–2022). At both sampling times, approximately one-fourth of all analyzed isolates had recombinant col1/cu genotypes. However, the frequency of MAT-1 isolates, which all have MAT-1 genes originating from O. ulmi, increased during this period. Differences in growth rate at 20, 26, and 30 °C revealed variations in the temperature response of isolates, which were affected by sampling time and mating type. The MAT-1 isolates were shown to grow more slowly than MAT-2 at the three temperatures tested. The advantage of MAT-2 was reflected in temporal differences in growth rate at resampled sites, particularly at lower temperatures. These results suggest that changes in the frequency of mating types in Croatia occurred between 2012 and 2022, accompanied by modifications in the pathogen’s response to temperature at the population level.

Global increases in connectivity have greatly accelerated the frequency of biological invasions across most of Earth’s ecosystems, including forests. Once invasive organisms become established in a naïve environment, they are difficult to eradicate or contain; thus, management strategies often focus on mitigating their impacts. As the use of chemical pesticides in forests is increasingly prohibited, biological control of pests and diseases has gained importance as an environmentally friendly alternative. Virus-mediated hypovirulence in the chestnut blight fungus Cryphonectria parasitica is one of the few successful examples of biological control of an invasive forest pathogen. However, experiments testing the stability of this system in situ are still missing. In this study, we conducted a field experiment in chestnut stands with naturally established hypovirulence in Switzerland, Croatia, and North Macedonia to evaluate the effectiveness of CHV1-mediated biocontrol of chestnut blight under different vegetative compatibility ( vc ) type population structures. Our results demonstrate that CHV1 is highly effective as a biological control agent against C. parasitica . Artificially initiated bark cankers of various vc types were rapidly infected by resident CHV1 strains, which significantly reduced canker growth and sporulation, thereby increasing the survival chances of the infected chestnut sprouts. Under field conditions, vegetative incompatibility barriers proved to be far less restrictive for virus transmission than predicted in vitro . Furthermore, our study demonstrates that the immigration of new fungal genotypes into existing cankers is an inherent component of the epidemiology of C. parasitica , which significantly contributes to the spread of CHV1. These results are particularly favourable for ensuring the success of hypovirulence-mediated biocontrol of chestnut blight in Europe. However, our conclusions cannot be automatically translated to genetically distant vc types from outside Europe, whose accidental introduction should be further avoided.