Background and Objective

Ash dieback caused by Hymenoscyphus fraxineus) has become to a serious threat for the survival of common ash (Fraxinus excelsior L.) in Europe. The disease is in progress steadily also in Mecklenburg-Vorpommern. Especially the economic and ecological damages are serious in the coastal region of Mecklenburg-Vorpommern, where ash is growing as main tree species at wet sites. Therefore, planting of ash is essential in the frame of close to nature forestry in Mecklenburg-Vorpommern. Propagation material with high resistance (determined genetically) against the fungus is needed in sufficient amount for reforestation of damaged sites.

It could be observed that a small number of ash trees (1 to 2 %) stays vital within severely damaged stands. Such trees will be selected, classified and grafted for establishing a seed orchard with a high genetic diversity.

In sub-project 2 the selected plus trees will be characterized phytopathologically and molecularly-genetically. Results are expected to genetical variability and diversity of ash in the northern part of Germany. Molecular markers will be developed and used for the detection of the fungus in plant material. Techniques for vegetative propagation (grafting, tissue culture) will be adapted and optimized. Ramets of plus trees will be propagated by grafting in sufficient amount for the future seed orchard. Tissue culture will be refined for propagation of root stocks with high resistance for graftings. A two-step procedure is used for testing the resistance of ramets (test with ascospores and infested wood chips). 

Analyses of paternity, planting of progeny tests and establishment of in vitro cultivation of adult plus trees are topics within the extension of the project time.

Approach

1. Development and adaptation of methods for the pytopatholgical and molecular genetical characterisation of ash
2. Identification by genotyping of single trees, investigation of diversity and genflow within and between stands of common ash, and comparison of genetic structure of infested and non-infested trees.
3. Characterisation of resistance status of selected plus trees.
4. Vegetative propagation of selected plus trees with high resistance for the establishment of a seed orchard.

Our Research Questions

Results

A protocol for the analysis of ash microsatellites comprising three primer sets with 16 primer pairs was developed and successfully applied. No clear population differences between the investigated ash stands in Mecklenburg-Vorpommern could be revealed.

Recultivation and further cultivation of different isolates of Hymenoscyphus fraxineus were performed. Detection of the pathogen in tissue of symptomatic leaves by real-time PCR was possible.

The design for the seed orchard in Tressow was elaborated. The orchard contains 126 clones with 1159 ramets.

Resistance of the grafts was tested by ascospores and a wood chip test. As a result of the resistance tests 26 plus tree clones were discarded due to increased disease susceptibility.

Fungal isolates from the Waldsieversdorf site did not prove to be unique in database matching with reference samples from other German sites.

Grafting methods used for vegetative propagation of plus trees were copulation and ‘goat foot’ grafting of scions with terminal buds in March in the greenhouse. In total, 3487 grafts were made from 152 plus trees. Plants from tissue culture (from resistant initial trees) and seedlings (49%) were used as rootstocks. The grafting success was 73%.

Different culture media were tested and modified for in vitro propagation of resistant clones. About 2150 plants from 23 clones were transferred to soil and some were used as grafting rootstocks.

Paternity analyses of five individual tree progenies identified several large full-sib families. These can be used in future projects for QTL analyses, for example.

Two progeny tests were created with 25 and 30 progenies, respectively. Survival was good at 95 and 88%, respectively.