Abstract
Chestnut plantations in South Tyrol (northern Italy) are an important element of the local landscape and provide additional income to the farmers. Chestnut blight, caused by Cryphonectria parasitica, is the most important disease of chestnut, responsible for the death of 99% of American chestnut trees at the beginning of the 20th century. The disease can be controlled by hypovirulence, characterized as a viral infection of C. parasitica by Cryphonectria hypovirus 1 (CHV-1), resulting in decreased virulence of the fungus against chestnut trees. Hypovuirulence may spread through hyphal anastomosis between two vegetatively compatible (VC) fungal strains. Increasing genetic diversity of the fungus, especially VC type and mating type variability, could impede the natural spread of hypovirulence in populations of C. parasitica. No information is available about the molecular genetic diversity of the fungus and the Hypovirus in South Tyrol, which may impede the biocontrol strategies of the fungus. Therefore, the aims of the thesis were the following; 1) to study the molecular genetic diversity and variability of C. parasitica present in South Tyrol based on vegetative incompatibility loci (vic), mating-type locus (MAT), DNA sequence analysis of the internal transcribed spacer region (ITS) and multilocus microsatellite typing. 2) To study the occurrence of CHV-1 infecting C. parasitica in South Tyrol and assess its molecular genetic variability. A total of 274 pure fungal isolates were obtained from diseased chestnut trees, representing all chestnut growing areas of South Tyrol. All molecular markers revealed a high diversity and variability of C. parasitica in South Tyrol such as VC type markers, microsatellite markers and ITS sequencing. Five out of six vic loci were polymorphic in the region with high overall variability. Twenty-three different VC types were present in South Tyrol, of which EU-2, EU-1 and EU-13 were the most dominant. Both mating types were found in the study area with a ratio close to 1:1, pointing to a potentially high rate of sexual reproduction. However, there were differences in mating-type ratios within some districts and many chestnut stands. ITS sequence analysis showed that 3 haplotypes of C. parasitica are present in South Tyrol and pointed to two different introduction events. Fifty-one multilocus genotypes were identified based on microsatellite loci, further demonstrating a high diversity and frequent sexual 3 reproduction in the region. Three genetic clusters of C. parasitica were identified in the region based on microsatellite DNA analysis, which supported the hypothesis of multiple introductions of the fungus to South Tyrol. A low degree of occurrence of hypovirulence (29.2%) highlighted a need for human-mediated application of virus-containing fungal strains in the region. All the sequences of CHV-1 belonged to the Italian subtype (I), which is the most widespread subtype in Europe. Some non-synonymous mutations in the RNA of CHV-1 demonstrated that some strains might be under negative selection, whereas others might be under positive selection. Phylogeographic analysis of CHV-1 showed that sequences from South Tyrol clustered closely with sequences of Swiss, Croatian, Slovenian and Bosnian CHV-1 strains. Based on this data, we recommend using hypovirulent strains of EU-2 and EU-1 as biocontrol agents in the region. These VC types strains could be particularly effective as biocontrol when contain locally identified CHV-1 haplotypes (such as HA1 and HA12) that could be under positive selection. A novel mycelium-blot immunoassay developed in this study might help detecting mycoviruses infecting C. parasitica, rapidly and accurately. This study will be useful to control C. parasitica in South Tyrol through biological methods and contributes to a better understanding of C. parasitica as well as CHV-1 genetic diversity and phylogeography in the region.