Gremmeniella abietina (Lagerberg) Morelet (anamorph Brunchorstia pinea (P. Karst) v Höhn) is a pathogenic fungus that produces severe damage in coniferous forests and reforestations, causing the death of the trees in Central and Northern Europe, North America and Japan. The pathogen was detected for first time in Spain in 1929 on Pinus pinaster (Aiton) and isolated in 1999 on Aleppo pine (Pinus halepensis (Mill)) stands in Palencia province. Although no epidemic outbreaks have been registered yet in Spain it causes serious damages to trees affected by the disease. The main symptoms observed consist of dry needles, crown defoliation, branches with some distortion of terminal twigs and eventual death of the trees. The control measures in other countries to prevent or reduce the expansion and incidence of the disease may include some silvicultural practices such as pruning branches or removing dead trees and the application of synthetic fungicides, but mostly as an emergency measure on nurseries. The need of management of the disease, together with the European recommendations of using non-chemical methods, make necessary to provide alternatives to the fight against the pathogen. Therefore, the main objective of the present thesis was to explore the use of alternative methods to control G. abetina infections. In order to achieve this objective, we performed four experiments in which we tested the use of several biological control agents (fungal endophytes, their filtrates, mitovirus-infected isolates) and the use of resistant hosts to prevent or reduce the infection.
The first two experiments were made with fungal endophytes and their filtrates because it was previously reported that these organisms were able to reduce or inhibit the growth of several pathogens by different modes of action. The fungal endophytes (first experiment) and their filtrates (second experiment) were applied to Aleppo pine seedlings as well as the G. abietina isolates were inoculated into the seedlings. Furthermore, to explore the mode of action of these biological control agents, the total phenol content of the plants after the infection was measured as an indicator of the activation of the plant defense system. The content of UV-absorving compounds of the filtrates was also measured to explore the possible presence of antibiotic substances. At the end of the experiment (6 months after infections) the necrosis produced by the advance of the pathogen was measured in all the seedlings. In order to verify the results, both experiments were repeated 3 weeks after the first inoculation took place. In the third experiment, we tested whether the presence of several mitoviruses (i.e., viruses associated with fungi from the genus Mitovirus and sometimes related to a decrease in the fitness of the pathogen) in the isolates of G. abietina, was able to modify the behavior and pathogenicity of the fungus both on in vitro conditions and on in vivo on Aleppo pine seedlings. Lastly, in the forth experiment, a provenance trial was performed with five different Spanish provenances of Aleppo pine in order to explore if any of them was resistant to the disease. The necrosis length produced by the advance of the pathogen was once again used as response variable. In addition, the concentration of two flavanone compounds as putative resistance indicator of the plants was measured and quantified.
The presence of all the endophytes used in our experiments as well as their filtrates significantly reduce the advance of G. abietina in the seedlings although sometimes the significance depended on the time of inoculation. When using the endophytes as biological control agents, the total phenolic content of the plant did not increase. Therefore, the mechanisms involved in that reduction may be the competition, the parasitism, the production of antibiotic compounds or a combination of them. Furthermore, low-molecular weight phenolic compounds could be detected in some but not all filtrates, suggesting that although the antibiosis was likely involved in that reduction, the endophytes probably use several modes of action. The results from the virus experiment showed that mitovirus-infected isolates presented larger mycelial growth than the mitovirus-free ones when at the fungi’s optimal growing temperature of 15 ºC. In the greenhouse experiment, larger necrosis lengths were observed in the plants inoculated with mitovirus-infected isolates, suggesting that the presence of viruses in G. abietina isolates could lead to a hypervirulence of the pathogen. Lastly, the Aleppo pine provenances tested in the fourth experiment presented different leves of susceptibility to the pathogen, in terms of necrosis and visual severity. The amount of naringenin flavanone was significantly different among provenances, suggesting that it is a possible indicator value for the resistance of the provenances.