Marine fungal community associated with standing plants of Spartina maritima (Curtis) Fernald

Tese de doutoramento, Biologia (Microbiologia), Universidade de Lisboa, Faculdade de Ciências, 2016The major purpose of this thesis was to complement the current knowledge regarding marine fungal communities and particularly those inhabiting Portuguese temperate salt marshes. Specifically, this study mainly intended to assess the species composition and diversity of the fungal communities associated with one of the most dominant macrophytes in these ecosystems, Spartina maritima (Curtis) Fernald, and to contribute to a better understanding of community dynamics and key ecological aspects of the fungi. The study was conducted in two geographically and physically distinct salt marshes, Castro Marim and Ria de Aveiro, where 195 mature, standing live plants were collected over a 2-year period (October 2010 to August 2012) from each study site. Each air-dried plant was separated into nine substrate categories according to the vegetative structure (leaf sheaths, stems and leaf blades) and physiological state of each structure (live, senescent and decaying). Identification of marine fungi was performed by two distinct, but complementary methods, i.e. direct observation of fungal structures (fruit bodies, spores and hyphopodia) and sequencing of the internal transcribed spacer regions of rDNA (ITS). The first method involved an individual observation of each substrate under dissecting- and light microscopes for detection of fungal structures; fungal taxa were morphologically identified using specific dichotomous keys for marine fungi. The vertical position and density of fruiting structures produced by each identified fungus was also recorded. The most frequent fungi were isolated in pure cultures by single spore method. Plant materials from the same substrate category, sampling period and study site were mixed and freeze-dried. Only the plant samples from the first sampling period were used for molecular identification of fungi. This second method involved DNA extraction of pure fungal isolates and plant samples, and amplification of the ITS region. Amplicons from plant samples were cloned in order to isolate individual amplicons of mixed PCR products. ITS sequences of the 1037 clones obtained from the plant samples were submitted to a restriction fragment length polymorphism analysis (RFLP); clones with different digestion profiles were sequenced. Phylogenetic analyses were performed with sequences of clones, fungal isolates and BLAST best-hits. A comparison between morphological and molecular methods revealed a general agreement in taxonomic assignments and representativeness of each fungus in the community, vegetative structure and study site. The combination of both methods was demonstrated to be crucial for a more realistic and accurate representation of the fungal community. Forty-five fungal taxa were recorded in S. maritima samples; 91% of these were filamentous ascomycetes, included in the Dothideomycetes and Sordariomycetes. The majority of the fungal species most frequently recorded in this study were previously described from other species of Spartina. Nevertheless, the studied fungal community also included other infrequent species that represent new records for the genus Spartina and/or S. maritima plants, e.g. Anthostomella spissitecta, Camarosporium roumeguerii, Ceriporia lacerata, Coniothyrium obiones, Cryptococcus mangaliensis, Decorospora gaudefroyi, Erythrobasidium hasegawianum, Halosarpheia trullifera, Leptosphaeria marina, Penicillium chrysogenum and Stagonospora haliclysta. The presence or absence of species in fungal communities may be related with intra- and interspecific differences in the physical structure and chemical composition of the host plants and/or macro and microenvironmental factors. Similarly to other grass-like plants, the results also demonstrated that the marine fungi are vertically distributed along standing plants of S. maritima. Moreover, the most frequent fungal taxa exhibited wide vertical distribution ranges, a high investment in the production of fruiting structures and were present during all the sampling period on senescent and decaying vegetative structures. The majority of these fungi were also found on live plant tissues, which indicated that these saprobic species might initiate the colonisation of plant substrates as endophytes. These findings suggested that the vertical distribution patterns, and occurrence and ecological role of most frequent fungi depend on the phase of plant life cycle and substrate availability, micro-environmental conditions of substrates and adaptation to submersion/exposure cycles, and potential fungal competitors. During the decay process of S. maritima, the obligate marine fungi Natantispora retorquens, Byssothecium obiones and Lulworthia sp.1 seem to be involved in the complete decomposition of lower leaf sheaths and stems; facultative marine fungi Mycosphaerella sp. I, of leaf blades; facultative marine fungi Phaeosphaeria halima and Stagonospora sp. 1, of upper standing leaves; and Buergenerula spartinae and Phaeosphaeria spartinicola, of all vegetative structures