European chestnut tree (Castanea sativa Mill.) has a great economical importance in Mediterranean Europe for its production of high quality wood and fruit. Portugal is the second largest producer of chestnuts, for which the region of Trás-os-Montes contributes for more than 85% of national production. In the last years, the decline of chestnut orchards from this region has been a consequence of severe diseases (ink disease, caused by Phytophthora cinnamomi and P.cambivora; blight-disease, caused by C. parasitica). Aware of the importance of this culture, team members have been devoted for the last 20 years to the understanding of the mechanisms underlying chestnut tree roots - pathogenic or mycorrhizal fungi interactions, and share a long tradition in collaborating in PhD and research projects. The PI team identified the fungus Hypholoma fasciculare from Trás-os-Montes (BraganÃ§a) chestnut orchards. Although this species is described as saprophytic trophic, preliminary results revealed that it can cause injuries to chestnut trees and displays strong antagonist activity against soil-borne fungi from orchard. A useful effect for orchard management could be found when H. fasciculare limits the growth and spreading of phytopathogenic fungi. However, as this fungus restricts the growth of beneficial fungi, its presence could be detrimental for the sustainability of the chestnut orchards. For example, the presence of ectomycorrhizic fungi (e.g. Pisolithus tinctorius) has been well-documented as having a beneficial effect on C. sativa orchards. To help the definition of the better management practices for chestnut orchards it would be fundamental to have the correct perception of the impact of H. fasciculare on the sustainability of chestnut orchards, either in plant itself or in the soil-borne fungal community. In this project, the characterization of C. sativa â€“ H. fasciculare interaction is proposed and concluding evidences for describing H. fasciculare as causing injuries in C. sativa plants are expected to be achieved. In vitro inoculation system will be used for studying morphological aspects of the interaction and inferring the pathogenic potential of H. fasciculare. The strong adhesion ability of hyphae to roots and the obstruction of vascular tissues are expected to be further characterized, and the chemical nature of the obstructing material is expected to be elucidated. These studies will be complemented by pot experiments to evaluate the conditions that promote pathogenicity: plant age, imposition of stressful conditions, the presence of other soil microorganisms and soil features. The elucidation of the pathogenic behaviour of H. fasciculare reveals to be of major importance, not only from the agronomic point of view, but also due to the ecological implications that it takes. H. fasciculare is already described as a biological agent to control root diseases caused by Armillaria sp. and field trials have already been performed to reduce A. ostoyae root disease. To our knowledge, H. fasciculare has never been described as causing plant injuries and any risk management study has been conducted before releasing or enhancing H. fasciculare population in target areas. To understand the high ability of this fungus to interact with chestnut tree root system, the involvement of hydrophobins in the adhesion of H. fasciculare will be studied. This will take advantage from a previous identified H. fasciculare hydrophobin DNA fragment, and will be performed by hydrophobin gene cloning and qRT-PCR analysis. Given the recognized beneficial effect of mycorrhization on C. sativa, the negative impact of H. fasciculare on the other soil-borne fungi will be also considered. For performing this, the below ground fungal diversity of chestnut soils will be determined for the first time, recurring to ITS-DGGE methods and making use of the population genetics expertise of project team members on the biodiversity analysis.
This project will have a positive social-economic impact has it will contribute to the reinvigoration of the most important culture of Trás-os-Montes, with the consequent economical and socio-cultural benefit for a depressed rural population. It is expected to contribute to wiser understanding of the impact of H. fasciculare on chestnut orchards and to help to define better management practices for increasing chestnut orchards sustainability.