Fusarium circinatum, the causal of pine pitch canker disease (PPC), is now considered among the most important pathogens of Pinaceae in the world. Although in Europe PPC is only established in the Iberian Peninsula, the potential endangered areas cover over 10 million hectares under the current host distribution and climatic conditions. It is therefore a priority to test the susceptibility of those species and their provenances, within Central and Northern Europe and find biological control agents (BCAs) against the disease. In this study, the susceptibility of Pinus sylvestris, P. mugo and Picea abies Romanian provenances to F. circinatum was tested using three inoculum doses. In parallel, the potential use of Trichoderma atroviride and Trichoderma viride as BCAs against F. circinatum was also tested. This study has demonstrated, for the first time, the susceptibility of P. mugo to F. circinatum. Likewise, the susceptibility of P. abies was also confirmed. The fact that the Romanian provenance of P. sylvestris has not been susceptible to F. circinatum suggests genetic resistance as a potential tool to manage the disease. This, together with the apparent effectiveness of Trichoderma species as BCAs, seems to indicate that an integrated management of the disease might be feasible.

23There is a growing necessity to replace chemical agents with ecofriendly materials, arising from 24 their impact on the environment and/or human health, which calls for the design of new broad-25 spectrum fungicides. In this work, chitosan oligomers (COs), propolis (Ps) and silver nanoparticles 26 (AgNPs) mixtures in solution were assessed to control the growth of different phytopathogenic fungi 27 and oomycetes in vitro. Binary solutions of COs-Ps and COs-AgNPs evinced the highest antifungal 28 effect against Fusarium circinatum and Diplodia pinea fungi, respectively, with a ca. 80% reduction 29 in their mycelial growth. The COs solution by itself also proved to be greatly effective against 30Gremmeniella abietina, Cryphonectria parasitica and Heterobasidion annosum fungi, causing a 31 reduction of 78%, 86% and 93% in their growth rate, respectively. Likewise, COs also attained a 32 100% growth inhibition on the oomycete Phytophthora cambivora. On the other hand, Ps inhibited 33 totally the growth of Phytophthora ×alni and Phytophthora plurivora. The application of AgNPs 34 reduced the mycelial growth of F. circinatum and D. pinea. However, the AgNPs in some binary and 35 ternary mixtures had a counter-productive effect on the anti-fungal/oomycete activity. In spite of the 36 fact that the anti-fungal/oomycete activity of the different treatments showed a dependence on the 37 particular type of microorganism, these solutions based on natural compounds can be deemed as a 38 promising tool for control of tree diseases. 39

Pine pitch canker (PPC), caused by Fusarium circinatum, is considered among the most important diseases affecting pines in many locations throughout the world. In Europe, F. circinatum is currently present in the Iberian Peninsula, posing a high risk of its spread into currently disease-free countries in Europe. In the present study, the susceptibility of Pinus sylvestris, Picea abies, and Larix decidua originating in the Czech Republic to F. circinatum was tested. Furthermore, the presence of asymptomatic yet infected seedlings was also checked. This study demonstrated the pathogenicity of F. circinatum to the Czech provenance of P. sylvestris, whereas Picea abies and Larix decidua proved to be tolerant. The reisolation of F. circinatum beyond the inoculation point demonstrated that this quarantine pathogen is able to infect the three conifers tested, giving rise to asymptomatic seedlings for at least eight and a half months. To our knowledge, this is the first study in which the presence of symptomless seedlings has been recorded in the genera Picea and Larix. This finding points out that the European legislation would fail to avoid the risk of new introductions via symptomless seedlings, since this legislation is only restricted to plants of the genus Pinus and the species Pseudotsuga menziesii.

Laccase enzymes (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) play a major role in the degradation of phenolic compounds such as lignin. They are common in fungi and have been suggested to participate in host colonization by pathogenic fungi. Putative mycoviruses have recently been isolated from the causal agent of pine pitch canker disease, Fusarium circinatum Nirenberg & O´Donell. In this study, the effects of single and double mycoviral infections on laccase activity, growth rate and pathogenicity were investigated in fourteen F. circinatum strains. Extracellular laccase activity was analyzed by the Bavendamm test, image processing and a spectrophotometric method. Mycelial growth, in vivo pathogenicity and seedling survival probability were also determined in Monterrey pine (Pinus radiata D. Don) seedlings. The findings showed that (i) mycelial growth of isolates from the same fungal population was homogeneous, (ii) the presence of mycovirus appears to increase the virulence of fungal isolates, (iii) co-infection (with two mycoviruses) caused cryptic effects in fungal isolates, and (iv) laccases embody a possible auxiliary tool in fungal infection. The prospects for biocontrol, the adaptive role of F. circinatum mycoviruses and the importance of laccase enzymes in host colonization are discussed.