Ljiljana Krstin

High diversity in populations of the introduced plant pathogen, Cryphonectria parasitica, due to encounters between genetically divergent genotypes.

The ascomycete fungus Cryphonectria parasitica is an aggressive introduced pathogen of sweet chestnut (Castanea sativa Mill.). It has spread throughout the chestnut‐growing areas of Europe, with higher diversity in the regions close to its first introduction and lower diversity in its expanding ranges in Europe. To reconstruct the invasion events that could explain the high diversity of C. parasitica in Croatia and Slovenia, 180 samples were genotyped using 11 sequence‐characterized amplified region markers. Eight of 11 loci were found to be polymorphic, and a total of 66 different haplotypes were identified. Bayesian clustering indicated the existence of two clusters, which suggests two separate introductions of C. parasitica in these regions. The first cluster is dominant in western parts of Croatia and Slovenia and the second in eastern and northern regions. The data analysis indicates that northern Italy was the first source of infection, with the subsequent introduction from south‐eastern Europe, which contributed significantly to the diversity of the C. parasitica populations tested. Most haplotypes were probably derived through sexual recombination between a few divergent haplotypes, which suggests that multiple introductions and sexual reproduction are important for the formation of genetically diverse C. parasitica populations.

Castanea sativa: genotype-dependent recovery from chestnut blight

In Lovran (coastal Croatia), a unique forest/orchard of evenly mixed grafted marrons and naturally growing nongrafted sweet chestnut trees exists. This old chestnut population has been devastated by chestnut blight, caused by an aggressive introduced pathogenic fungus, Cryphonectria parasitica. However, initial observations indicated recovery of naturally growing chestnut trees in that area, mediated by Cryphonectria-associated hypovirus (Cryphonectria hypovirus 1 (CHV-1)). Such recovery was not observed on grafted trees. Genotyping both, we confirmed the clonal origin of the grafted ones—marrons. No significant difference was observed between fungal strains isolated from naturally growing trees and the ones from marrons regarding fungal vegetative compatibility types or the prevalence of CHV-1. A strong correlation was observed between the types of canker: active/deep-expanding versus healing callus or superficial necrosis and the absence or presence of CHV-1 in the fungal isolates, sampled from naturally growing trees (Spearman rho 0.686, p value 7.81 × 10−5, Kendall tau 0.686, p value 5.18 × 10−7). Such correlation was not observed on marrons (Spearman rho 0.236, p value 0.235, Kendall tau 0.236, p value 0.084), because, unexpectedly, active/deep-expanding cankers were often associated with hypovirulent fungal isolates. These data indicate that the lack or unequal distribution of naturally occurring hypovirulence were not the cause of substantial marron decay in Lovran. Ecological and age-dependant differences were ruled out because all sampled trees are growing in close proximity and are of similar age. The results imply that the marron genotype is especially vulnerable and its ability to recover is limited even when the hypovirulent strain of the fungus is present in the canker.

Biological control of chestnut blight in Croatia: an interaction between host sweet chestnut, its pathogen Cryphonectria parasitica and the biocontrol agent Cryphonectria hypovirus 1

BACKGROUND Chestnut blight, caused by the fungus Cryphonectria parasitica, is a severe chestnut disease that can be controlled with naturally occurring hypoviruses in many areas of Europe. The aim of this research was to measure the effect of different Cryphonectria hypovirus 1 (CHV1) strains on the growth of the fungal host and select strains that could potentially be used for human-mediated biocontrol in forests and orchards, and to investigate whether and how chestnut-fungus-virus interactions affect the development and growth of the lesion area on cut stems. RESULTS Two Croatian CHV1 strains (CR23 and M56/1) were selected as potential biocontrol agents. The sequencing of CHV1/ORF-A showed that both of these virus strains belonged to the Italian subtype of CHV1. In vitro transfection of selected virus strains from hypovirulent to genetically diverse virus-free fungal isolates and subsequent inoculation of all virus/fungus combinations on stems of genetically diverse sweet chestnut trees revealed that Croatian virus strain CR23 had an equally hypovirulent effect on the host as the strong French strain CHV1-EP713, while M56/1 had a weaker effect. Furthermore, it was shown that in some cases the same hypovirus/fungus combinations induced various degrees of canker development on different chestnut genotypes. CONCLUSION Some CHV1 strains belonging to the Italian subtype have similar hypovirulent effects on C. parasitica to those belonging to the French subtype. Furthermore, chestnut susceptibility and recovery could be influenced by the response of chestnut trees to particular hypovirulent C. parasitica isolates, and virus-fungus-chestnut interactions could have significant implications for the success of chestnut blight biocontrol.

Cryphonectria hypovirus 1-Induced Changes of Stress Enzyme Activity in Transfected Phytopathogenic Fungus Cryphonectria parasitica

Cryphonectria parasitica is a phytopathogenic fungus introduced from Eastern Asia to North America and to Europe, where it causes chestnut blight, a devastating disease of chestnut trees. The disease can be biologically controlled utilising the mycovirus Cryphonectria hypovirus 1 (CHV1), which changes the physiology of the host, reducing its virulence towards chestnut. We measured fungal growth in vitro and activities of glutathione S-transferase, catalase and superoxide dismutase, enzymes involved in oxidative stress response, to elucidate the effects of CHV1 infection on the host. Six CHV1 strains of different subtypes and three fungal isolates were used in different combinations to better represent natural conditions, where higher genetic diversity of both fungus and virus is expected. The infection with different CHV1 strains decreased in vitro growth rate of infected fungal isolates and increased activity of their stress enzymes in most of the studied fungus/virus combinations, indicating increased oxidative stress following CHV1 infection. All our field CHV1 strains belong to the Italian subtype, but while strain M56-1 had equal or even stronger effect on its fungal host than prototypic strain EP713 of French subtype F1, strain B11 had no effect. Thus, the severity of the observed effects depended on a particular virus strain, fungal isolate, and the combination of the two, rather than solely on the virus subtype. Since previous research showed discordance between accumulation of mRNA and stress-related proteins in CHV1 infected C. parasitica, our results emphasise the importance of enzymes’ activity measurements as an invaluable extension of transcriptomic and proteomic analyses.

Changes in Cryphonectria parasitica populations affects natural biological control of chestnut blight

Invasive species, especially plant pathogens, have a potential to completely eradicate native plant species and remodel landscapes. Tripartite interactions among sweet chestnut (Castanea sativa), chestnut blight-causing invasive fungus Cryphonectria parasitica, and hyperparasitic virus Cryphonectria hypovirus 1 (CHV1) were studied in two populations. The number of different vegetative compatibility (vc) types of C. parasitica more than doubled over the 10 years, while the hypovirulence incidence dropped in one population and slightly increased in the other one. Over the course of our 3-year monitoring experiment, the prevalence of hypovirulent isolates obtained from monitored cankers increased slowly (i.e., more hypovirulent isolates were being obtained from the same cankers over time). Within studied cankers, considerable changes in vc type and CHV1 presence were observed, indicating a highly dynamic system in which virulent and hypovirulent mycelia, sometimes of discordant vc types, often appeared together. The increase in hypovirulence prevalence did not have any observable curative effect on the cankers and, occasionally, reactivation of healed cankers by new, virulent C. parasitica isolates was observed. Both short- and long-term observations and revalidation of the infected plant populations are necessary to accurately estimate disease progress and formulate an adequate disease management strategy.