Fusarium circinatum – host interactionecological and molecular aspects of the pathogenic and endophytic association

Resumo

Fusarium circinatum Nirenberg & O’Donnell, the causal agent of pitch canker disease, is one of the most important pathogens of conifers worldwide, affecting at least 57 species of Pinus, and causing important economic and environmental losses in nurseries and forest plantations. The fungus has been traditionally considered exclusively a pathogen of conifers, not taking into account the possibility of other symbiotic relationships with the host. The fungus was recently detected living as an endophyte in grasses causing no apparent damage. The main objective of this dissertation is to study ecological and molecular aspects of F. circinatum and its host association, exploring both endophytic and pathogenic lifestyles. In Chapter 4 we explored the non-symptomatic host range of the fungus in a plantation of Pinus radiata with symptoms of pine pitch canker (PPC) disease and we also tested whether this non-symptomatic host may act as a reservoir of inoculum. We detected the fungus in five species of dicot families (Asteraceae, Lamiaceae, Rosaceae), in addition to two species in the Poaceae. The fungus was found in the aerial part of non-symptomatic hosts, so we describe F. circinatum as an endophyte that is mainly transmitted by spores through the air. Vertical transmission may also occur at least in one species, Hypochaeris radicata, since fungus was detected in seeds of these plants. Endophytes frequently sporulate when infected host tissue dies, and we proved that pine seedlings can be infected from senescent tissue of non-symptomatic hosts colonized by the fungus under laboratory conditions. This endophytic stage of F. circinatum is not exclusive of herbaceous plants, since the existence of a latent stage within pine host tissue has previously been cited. However, studies on this cryptic phase in the disease life cycle have only been focused on the host aerial part but not on the roots. We analysed the presence of the F. circinatum in roots of non-symptomatic mature trees in Pinus pinaster and P. radiata plantations, where the pathogen is known to be causing canker symptoms. The fungus was isolated from roots of non-symptomatic P. radiata trees in a higher frequency than from roots of symptomatic trees. Host susceptibility seems to be involved in the presence of the pathogen in roots because it was not isolated from any roots in the P. pinaster plantation, pine species reported to have moderate resistance to the pathogen. An analysis of microsatellite markers showed a unique haplotype in F. circinatum population regardless of whether the isolates origin was pine cankers, symptomless pine roots or non-symptomatic herbaceous plants. Thus, the same haplotype can adopt a pathogenic or endophytic lifestyle. Indeed, all isolates resulted pathogenic to pines by artificial inoculations. These results show the potential risk of spread of F. circinatum across non-symptomatic species that serve as reservoirs of inoculum and provide insights into the epidemiology of pitch canker disease. In addition, root infection is a relevant factor in the disease cycle, and this finding needs to be considered hereafter in management of the PPC disease. In spite of the importance of the disease, knowledge of the molecular basis of the pathogenic interaction of F. circinatum with Pinus trees is limited. P. pinaster is an ecological and economical important species in the Mediterranean area and, although natural infections have been reported, the species have shown a moderate resistance to the pathogen. For these reason we study F. circinatum-P.pinaster interaction at a molecular level, trying to determine which mechanism are strongly involved in plant resistance and pathogen virulence. For this purpose, in the absence of a reference genome, we generated a high quality de novo P. pinaster transcriptome assembly (Chapter 5), used for downstream gene expression analyses. By a dual RNA-sequencing approach, we determined the expression profiling of both organisms during the interaction at 3, 5 and 10 days post-inoculation (Chapter 6). The moderate resistance that P. pinaster showed at the early time points, may be explained, at least in part, by the early activation of defence response based on the induction of pathogenesis-related proteins and the activation of complex phytohormone signalling that involves crosstalk between salicylic acid, jasmonic acid and ethylene. Moreover, we hypothesize the key steps where the pathogen could be manipulating host phytohormone balance to its own benefit, contributing to host susceptibility. Upon examination of the pathogen transcripts, we propose that F. circinatum prevents salicylic acid biosynthesis from the chorismate pathway by the synthesis of isochorismatase family hydrolase (ICSH) genes, perturbs ethylene homeostasis in the host by expression of genes related to ethylene biosynthesis, and could be blocking jasmonic acid signalling by COI1 suppression. In summary, this work present a broader repertoire of interaction of F. circinatum with its hosts and evidence the importance that an endophytic stage of the pathogen may have in the disease lifecycle, and therefore, for disease management. Indeed, we explore the molecular mechanism involved in the pathogenic interaction of F. circinatum with P. pinaster, analysing which processes are responsible for plant defence and pathogen virulence, providing new knowledge in mechanisms by which PPC is caused.