Colletotrichum truncatum tem sido amplamente estudado como fitopatógeno da soja. No entanto, diferentes isolados possuem diferentes graus de agressividade, podendo, inclusive, serem encontrados em plantas aparentemente sadias. Assim, primeiramente objetivou-se comparar isolados de C. truncatum obtidos de plantas assintomáticas e sintomáticas de soja, a fim de identificar características que os diferenciassem. Obtiveram-se 17 isolados de plantas assintomáticas e três de plantas doentes. Com os testes de patogenicidade, verificou-se que alguns isolados obtidos de plantas assintomáticas podem ser endofíticos e que outros causaram sintomas na parte aérea, com severidade variável, e afetaram a germinação e a emergência de plântulas obtidas a partir de sementes inoculadas. Na análise concatenada dos genes ITS, GAPDH, HIS3 e do gene ß-tub verificou-se que isolados caracterizados como hipovirulentos e endofíticos foram alocados em um mesmo clado, assim como os mais agressivos também se agruparam. Na análise do marcador molecular IRAP não houve diferenças. Em meio SNA isolados mais agressivos tiveram menores taxas de crescimento, porém, esporulação em maiores quantidades, ao contrário, avirulentos e hipovirulentos. A germinação de conídios dos isolados agressivos foi maior quando comparada à dos isolados endofíticos e hipovirulentos, assim como a porcentagem de conídios formando apressório. Em relação à atividade enzimática in vitro, não foi possível a diferenciação dos isolados com diferentes graus de patogenicidade utilizando-se sistema API-ZYM® e todos os isolados obtidos produziram as enzimas esterase, esterase-lipase, fosfatase ácida e a naftol-AS-BI- fosfohidrolase. Para a atividade de pectina-liase, isolados mais agressivos produziram menos pectina liase que os não agressivos. A partir da identificação dos dois estilos de vida, selecionou-se um isolado endofítico e um patogênico para verificar a capacidade de C. truncatum endofítico ativar o sistema de defesa de plantas de soja e conferir proteção contra a antracnose e mofo branco (Sclerotinia sclerotiorum). Para tanto, plantas de soja foram inoculadas com os isolados endofítico patogênico de C. truncatum, separadamente. Um outro tratamento consistiu da pré-inoculação do endofítico, seguido do patogênico. Avaliou-se a atividade de proteínas relacionadas à patogênese - PR-proteínas (peroxidases - POX e fenilalanina amônia-liase-FAL) e da quantidade de peróxido de hidrogênio (H2O2). Plantas de soja foram pré-inoculadas com o endofítico seguido de inoculação de S. sclerotiorum após cinco dias, sendo avaliada a severidade do mofo branco após 10 dias da inoculação do mesmo. Plantas pré-inoculadas com C. truncatum endofítico e desafiadas com o C. truncatum patogênico tiveram aumento da atividade de POX e da FAL e da quantidade de H2O2. Quando inoculados separadamente, endofítico e patogênico não foram capazes de aumentar a atividade ou quantidade dessas moléculas. Demonstrou-se redução de mais de 88% na severidade do mofo-branco em plantas de soja pré-inoculadas com isolado endofítico. A redução da doença pode ser devido a capacidade dessas plantas em aumentar a resposta de defesa mais rapidamente e em níveis maiores do que quando expostas ao endofítico isoladamente. / Colletotrichum truncatum, the causal agent of anthracnose of soybean: a comparison of isolates obtained from asymptomatic and symptomatic plants Colletotrichum truncatum has been widely studied as soybean pathogen. However, different isolates have different degrees of aggressiveness, and it can even be found in apparently healthy plants. Thus, the first objective was to compare isolates of C. truncatum from asymptomatic and symptomatic soybean plants, in order to identify characteristics that differentiate them. Seventeen asymptomatic plant isolates and three diseased plants were obtained. From the pathogenicity tests, it was verified that some isolates obtained from asymptomatic plants could be endophytic and others caused symptoms in the aerial part, with variable severity, and affected the germination and emergence of seedlings obtained from inoculated seeds. In the concatenated analysis of the genes ITS, GAPDH, HIS3 and ß-tub gene, it was verified that the isolates characterized as hypovirulent and endophytic were allocated in the same clade, as well as the more aggressive were also grouped. In the analysis of the molecular marker IRAP there were no differences. In the SNA (Synthetic - Poor in Nutrient - Agar) medium, the aggressive isolates had the lowest growth rates, however, they had higher sporulation. The endophytic and hypovirulent ones had higher rates of mycelial growth and less sporulation in vitro. The germination of conidia and the percentage of conidia forming appressory of the aggressive isolates was higher when compared to the endophytic and hypovirulent isolates. In relation to the enzymatic activity in vitro, it was not possible to differentiate the isolates with different degrees of pathogenicity using the API-ZYM® system. Furthermore, all the isolates obtained produced the enzymes esterase, esterase-lipase, acid phosphatase and naphthol-AS- BI-phosphohydrolase. For pectin-lyase activity, isolates that are more aggressive produced less lyase pectin than non- aggressive pectin. From the identification of the two lifestyles, an endophytic and pathogenic isolate were selected to verify the ability of C. truncatum to activate the soybean defense system and to protect soybean plants against anthracnose and white mold (Sclerotinia sclerotiorum). For that, soybean plants were inoculated with an endophytic, a C. truncatum pathogenic, and an endophytic followed by the inoculation of the pathogen after 5 days. These plants were evaluated for the activity of proteins related to the pathogenesis - PR-proteins (peroxidases - POX and phenylalanine ammonia-lyase-FAL), the amount of hydrogen peroxide (H2O2) and malondialdehyde (MDA). Soybean plants were pre-inoculated with the endophytic followed by inoculation of S. sclerotiorum after five days, and the severity of the white mold was evaluated after 10 days of inoculation. Plants pre-inoculated with endophytic C. truncatum and challenged with pathogenic C. truncatum had increased POX and FAL activity and the amount of H2O2. When inoculated separately, endophytic and pathogenic were not able to increase the activity or quantity of these molecules. There was a reduction of more than 88% in the severity of white mold in soybean plants preinoculated with endophytic isolate. The reduction of the disease may be due to the ability of these plants to increase the defense response more rapidly and at higher levels than when exposed to the endophyte alone.

Plant roots host a wide spectrum of endophytic fungi ranging from parasites through neutralistic fungi to mutualistic mycorrhizal fungi. In most of terrestrial ecosystems, these groups of symbiotic fungi are well documented. However, much less is known about fungal endophytes of aquatic plants, especially the only group of submerged marine plants, seagrasses. We focused on roots of the seagrass species Posidonia oceanica which is a Mediterranean endemite. Its roots hosted an abundant presence of endophytic fungi. We examined the roots using optical and electron microscopy in order to study its morphology. We isolated the mycobionts and determined their taxonomic classification. A specific symbiosis of P. oceanica roots and dark coloured septate fungus has been recorded all over a vast area of the Mediterranean spreading from southern Spain to south Turkey. The fungus forms distinctive and typical structures: superficial hyphae occasionally forming hyphal sheaths or dense hyphal nets, intraradical hyphae colonizing extracellular space and, occasionally, primary cortex cells, and finally intracellular microsclerotia. The colonization pattern of the fungi resembles dark septate endophytes (DSE), group of fungi commonly found in roots of both terrestrial and freshwater plants. In our following studies, we...

Seagrasses are the only group of submerged plants that are permanently growing in marine environment. They play an important role in the sea bottom ecosystem. Seagrasses are primary producers capable of accumulation and deposition of carbon. They influence water flow at the sea bottom and form symbioses with variety of organisms. Our knowledge of symbiotic interactions of seagrasses is still limited even though several studies of the topic have been carried out in recent years. Unlike most of terrestrial plants, seagrasses are generally considered as plants that do not form any specific associations with mycorrhizal or endophytic fungi. Surprisingly, we have discovered a novel fungal endophytic association in roots of Mediterranean endemic seagrass species Posidonia oceanica (L.) Delile. Morphology of this symbiotic fungi strongly resembles common symbiotic fungi of terrestrial plants, dark septate endophytes (DSE). We sampled roots of P. oceanica in large area of the Mediterranean from southeastern Spain to Albania and described range and taxonomical classification of the endophyte using microscopy, in vitro cultivation and molecular determination. Roots of P. oceanica in whole area of study are colonized by mere two endophytic fungal species. Over 90 % of the fungal symbionts belong to a single...

En esta tesis doctoral se estudia el modo de vida tritrófico del hongo nematófago Pochonia chlamydosporia utilizando técnicas "multiómicas". Pochonia chlamydosporia (= Metacordyceps chlamydosporia) (Goddard) Zare y Gams es un hongo nematófago usado para el control de nematodos agalladores de la raíz (Meloidogyne spp.) (Forghani and Hajihassani, 2020), entre otros. P. chlamydosporia se distribuye por todo el mundo y tiene un modo de vida tritrófico, pudiendo también adoptar estilos de vida endófito y saprófito. El mecanismo que utiliza P. clamydosporia para infectar huevos de nematodo comprende la desacetilación de la quitina de su pared celular a quitosano para facilitar su degradación por quitosanasas (Aranda-Martinez et al., 2016). El quitosano es un biopolímero derivado de la quitina que también se encuentra en el exoesqueleto de artrópodos y crustáceos. El genoma de P. chlamydosporia codifica un elevado número de quitosanasas, gracias a las cuales es resistente a quitosano y puede utilizarlo como fuente de nutrientes (Palma-Guerrero et al., 2010). Ambos pueden combinarse para el control de plagas. En este trabajo de tesis doctoral se pretende estudiar mediante metabolómica, transcriptómica y genómica el modo de vida tritrófico de P. chlamydosporia añadiendo quitosano, para determinar los mecanismos de interacción del hongo en ese entorno. En último término, se pretende sentar las bases para desarrollar un sistema para reducir plagas y enfermedades de forma sostenible.