Browsing by Author "Oliveira, Helena"
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- Agrobacterium-mediated transformation and insertional mutagenesis in Colletotrichum acutatum for investigating varied pathogenicity lifestylesPublication . Oliveira, Helena; Talhinhas, Pedro; Martins, João Neves; Muthumeenakshi, S.; Sreenivasaprasad, S.Colletotrichum acutatum is a cosmopolitan pathogen causing economically important diseases known as anthracnose on a wide range of hosts. This fungus exhibits varied pathogenicity lifestyles and the tools essential to understand the molecular mechanisms are still being developed. The transformation methods currently available for this species for gene discovery and functional analysis involve protoplast transformation and are laborious and inefficient. We have developed a protocol for efficient Agrobacterium tumefaciens-mediated transformation (ATMT) of C. acutatum. Using this protocol we were able to transform C. acutatum isolates belonging to different genetic groups and originating from different hosts. The transformation efficiency was up to 156 transformants per 104 conidia, with[70% transformants showing single location/single copy integration of T-DNA. Binary vector pBHt2-GFP was constructed, enabling green fluorescence protein tagging of C. acutatum strains, which will be a useful tool for epidemiology and histopathology studies. The ATMT protocol developed was used to identify putative pathogenicity mutants, suggesting the applicability of this technique for rapid generation of a large panel of insertional mutants of C. acutatum leading to the identification of the genes associated with the varied lifestyles.
- Caracterização de endófitos da videira pertencentes ao complexo Epicoccum nigrum e sua interação com fungos do lenho da videiraPublication . Coletto, Tatiana Santos; Nascimento, Teresa; Cabral, Ana; Oliveira, HelenaEndófitos do género Epicoccum são frequentemente isolados do lenho da videira, associados ou não a fungos patogénicos, designadamente a Fomitiporia mediterranea, Phaeomoniella (Pa.) chlamydospora, Phaeoacremonium (Pm.) minimum (sin. Pm. aleophilum), bem como a fungos “Botryosphaeriaceae”, mas desconhece-se o seu efeito em relação aos fungos que coabitam com esses endófitos. O presente estudo teve como objetivo avaliar a variabilidade de uma coleção de endófitos, do género Epicoccum (Epic), obtida a partir do lenho da videira e contribuir para a melhor compreensão das interações que estabelecem in vitro com fungos patogénicos do lenho da videira. Com base em características culturais e na sequenciação de parte do gene da β-tubulina (TUB2), os isolados foram agrupados em quatro grupos dentro do complexo E. nigrum, que revelou ser um complexo de espécies crípticas. Para avaliação das interações Epic versus fungos do lenho da videira, a coleção foi previamente testada contra um fungo “Botryosphaeriaceae”, tendo o isolado Epic 2 surgido no grupo dos que revelaram atividade antagonista significativamente mais elevada (α= 0,05). Epic 2 inibiu também significativamente o crescimento micelial dos fungos do complexo da esca, Pa. chlamydospora (45,8%), F. mediterranea (42,7%) e Pm. minimum (18,8%). Discutem-se os mecanismos de interação entre endófitos e fungos patogénicos do lenho da videira, bem como o potencial apresentado por Epic 2 como antagonista
- Characteristics of portuguese strains of Agrobacterium tumefaciens isolated from grapevine and stone fruit treesPublication . Oliveira, Helena; Nascimento, TeresaProspecções efectuadas em vinhas e pomares no País revelaram a presença do agente causal do tumor bacteriano, Agrobacterium tumefaciens. Com base no estudo das características morfológicas, culturais, bioquímicas e de patogenicidade, quarenta isolamentos de Agrobacterium spp. foram identificados e separados nos respectivos biótipos. A maioria das estirpes isoladas apresentaram características inerentes aos biótipos 1 e 2. No caso da videira, foi também detectada a presença da bactéria no translocado xilémico, o que evidencia a natureza sistémica desta doença
- Characterization of the wood mycobiome of Vitis vinifera in a vineyard affected by Esca. Spatial distribution of fungal communities and their putative relation with leaf symptomsPublication . Del Frari, Giovanni; Gobbi, Alex; Aggerbeck, Marie Ronne; Oliveira, Helena; Hansen, Lars Hestbjerg; Ferreira, Ricardo BoavidaEsca is a disease complex belonging to the grapevine trunk diseases cluster. It comprises five syndromes, three main fungal pathogenic agents and several symptoms, both internal (i.e., affecting woody tissue) and external (e.g., affecting leaves and bunches). The etiology and epidemiology of this disease complex remain, in part, unclear. Some of the points that are still under discussion concern the sudden rise in disease incidence, the simultaneous presence of multiple wood pathogens in affected grapevines, the causal agents and the discontinuity in time of leaf symptoms manifestation. The standard approach to the study of esca has been mostly through culture-dependent studies, yet, leaving many questions unanswered. In this study, we used Illumina R next-generation amplicon sequencing to investigate the mycobiome of grapevines wood in a vineyard with history of esca. We characterized the wood mycobiome composition, investigated the spatial dynamics of the fungal communities in different areas of the stem and in canes, and assessed the putative link between mycobiome and leaf symptoms. An unprecedented diversity of fungi is presented (289 taxa), including five genera reported for the first time in association with grapevines wood (Debaryomyces, Trematosphaeria, Biatriospora, Lopadostoma, and Malassezia) and numerous hitherto unreported species. Esca-associated fungi Phaeomoniella chlamydospora and Fomitiporia sp. dominate the fungal community, and numerous other fungi associated with wood syndromes are also encountered (e.g., Eutypa spp., Inonotus hispidus). The spatial analysis revealed differences in diversity, evenness and taxa abundances, the unique presence of certain fungi in specific areas of the plants, and tissue specificity. Lastly, the mycobiome composition of the woody tissue in proximity to leaves manifesting ‘tiger stripes’ symptoms of esca, as well as in leaf-symptomatic canes, was highly similar to that of plants not exhibiting any leaf symptomatology. This observation supports the current understanding that leaf symptoms are not directly linked with the fungal communities in the wood. This work builds to the understanding of the microbial ecology of the grapevines wood, offering insights and a critical view on the current knowledge of the etiology of esca
- Control of grapevine wood fungi in commercial nurseriesPublication . Oliveira, Helena; Rego, Cecilia; Nascimento, Teresa; Cabral, Ana; Silva, Maria JoséPrevious surveys conducted in commercial nurseries found that different wood fungi, namely Cylindrocarpon spp., Botryosphaeriaceae, Phomopsis viticola and Phaeomoniella chlamydospora infect grapevine cuttings. Two fi eld trials were carried out to evaluate the effectiveness of cyprodinil + fl udioxonil, pyraclostrobin + metiram, fl udioxonil and cyprodinil to prevent or reduce natural infections caused by such fungi. Rootstock and scion cuttings were soaked in fungicidal suspensions for 50 min prior to grafting. After callusing, the grafted cuttings were planted in two commercial fi eld nurseries with and without a previous history of grapevine cultivation. After nine months in the nursery, the plants were uprooted and analysed for the incidence and severity of the wood fungi. Plants uprooted from the fi eld without a previous history of grapevine cultivation were generally less strongly infected by wood fungi. Under this condition, only the mixture cyprodinil + fl udioxonil simultaneously reduced the incidence of Cylindrocarpon and Botryosphaeriaceae fungi, as well as the severity of Cylindrocarpon infections. Treatments did not produce signifi cant differences in the incidence and severity of P. viticola, and Pa. chlamydospora. For plants grown in the fi eld with a grapevine history, all fungicides except cyprodinil signifi cantly reduced the incidence and severity of Cylindrocarpon fungi. Also, the incidence and severity of Botryosphaeriaceae pathogens were signifi cantly decreased both by cyprodinil + fl udioxonil and by cyprodinil. No signifi cant differences were noticed for P. viticola incidence and severity, and Pa. chlamydospora was not detected again. These results suggest that the practice of soaking grapevine cuttings in selected fungicides prior to grafting signifi cantly reduces Cylindrocarpon spp. and Botryosphaeriaceae infections, thus improving the quality of planting material.
- Correlations between disease severity, glucosinolate profiles and total phenolics and Xanthomonas campestris pv. campestris inoculation of different BrassicaceaePublication . Aires, Alfredo; Dias, Carla S.P.; Carvalho, Rosa; Oliveira, Helena; Monteiro, António A.; Simóes, Manuel Vieira; Rosa, Eduardo A.S.; Bennett, Richard N.; Saavedra, Maria JoséMany Brassicaceae species are economically important crops and Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot, is considered one of the most important necrotrophic plant bacterial diseases occurring worldwide on these and many other crops. Therefore identifying resistance mechanisms and genes is crucial. Researchers continue to investigate the role of phytochemicals (plant secondary metabolites) in protecting plants against diseases and pathogens. Glucosinolates (GLS), and more specifically their hydrolysis products, are known to have various biological effects including antimicrobial activity. From the positive results of initial in vitro studies with Xcc and other pathogenic bacteria new experiments were designed to evaluate the possible in planta role of GLS, and also phenolics, in the interaction with Xcc. The in planta studies, with various Brassicaceae seedlings, have shown a correlation between GLS profiles, and therefore the subsequent hydrolysis products, and the inhibition of Xcc growth. There were no significant correlations between Xcc infection and total phenolics. Positive correlations were found between specific and total GLS contents and the severity of disease. Further in vitro and in planta studies need to be performed to evaluate the role of GLS and other defense mechanisms in Xcc and other important bacterial infections of Brassicaceae crops.
- Cylindrocarpon root rot: multi-gene analysis reveals novel species within the Ilyonectria radicicola species complexPublication . Cabral, Ana; Groenewald, Johannes; Rego, Cecília; Oliveira, Helena; Crous, Pedro W.Abstract Ilyonectria radicicola and its Cylindrocarpon-like anamorph represent a species complex that is commonly associated with root rot disease symptoms on a range of hosts. During the course of this study, several species could be distinguished from I. radicicola sensu stricto based on morphological and culture characteristics. DNA sequence analysis of the partial β-tubulin, histone H3, translation elongation factor 1-α and nuclear ribosomal RNA-Internal Transcribed Spacer (nrRNA-ITS) genes were employed to provide further support for the morphological species resolved among 68 isolates associated with root rot disease symptoms. Of the various loci screened, nrRNA-ITS sequences were the least informative, while histone H3 sequences were the most informative, resolving the same number of species as the combined dataset across the four genes. Within the Ilyonectria radicicola species complex, 12 new taxa are delineated occurring on a diverse range of hosts, the most common being Cyclamen, Lilium, Panax, Pseudotsuga, Quercus and Vitis.
- Epicoccum layuense a potential biological control agent of esca-associated fungi in grapevinePublication . Del Frari, Giovanni; Cabral, Ana; Nascimento, Teresa; Ferreira, Ricardo Boavida; Oliveira, HelenaEpicoccum is a genus of ascomycetes often associated with the mycobiome of grapevines (Vitis vinifera). Epicoccum spp. are found in the soil, phyllosphere, as well as in the wood, where they interact both with the plant and with other endophytes and pathogens. Wood pathogens involved in the esca disease complex, a grapevine trunk disease, are particularly concerning in viticulture, as current control strategies have proven unsatisfactory. This study investigated the interaction among Epicoccum spp. and three esca-associated fungi, with the aim of establishing whether they are suitable candidates for biological control.A screening conducted in vitro, by means of dual culture, revealed that all tested Epicoccum spp. inhibited the growth of pathogens Phaeomoniella chlamydospora and Fomitiporia mediterranea, while only some of them inhibited Phaeoacremonium minimum. Epicoccum layuense E24, identified as the most efficient antagonist, was tested in rooted grapevine cuttings of cultivars Cabernet Sauvignon and Touriga Nacional, under greenhouse conditions, against P. chlamydospora and P. minimum. This study revealed that the inoculation of E. layuense E24 produced a successful colonization of the wood of grapevines; in addition it did not impair the growth of the plants or induce the appearance of symptoms in leaves or in wood. Moreover, grapevines colonized by E. layuense E24 showed a considerable decrease in the wood symptomatology caused by the inoculated pathogens (by 31–82%, depending on the pathogen/grapevine cultivar), as well as a reduction in their frequency of re-isolation (60–74%).Our findings suggest that E. layuense E24 is a promising candidate for its application in biological control, due to its antagonistic interaction with some escaassociated fungal pathogens
- Epidemiology, histopathology and aetiology of olive anthracnose caused by Colletotrichum acutatum and C. gloeosporioides in PortugalPublication . Talhinhas, P.; Mota-Capitão, C.; Martins, S.; Ramos, Ana Paula; Neves-Martins, J.; Guerra-Guimarães, L.; Várzea, V.; Silva, M.C.; Sreenivasaprasad, S.; Oliveira, HelenaAnthracnose is an important disease affecting mature olive fruits, causing significant yield losses, and poor fruit and oil quality. In Portugal, high anthracnose incidence was recorded during 2003–2007 with 41%of 908 orchards surveyed displaying disease symptoms. In another 14% of the orchards, the pathogen was recorded in symptomless plants. Disease severity was on average 36%, frequently reaching 100%. In Portugal, anthracnose is endemic to neglected orchards of susceptible cultivars, but under favourable conditions it can also severely affect less susceptible cultivars. Pathogens were genetically heterogeneous, with Colletotrichum acutatum genetic group A2 as the most frequent (80%), followed by group A4 (12%) and group A5 along with C. gloeosporioides (3–4%), while groups A3 and A6 of C. acutatum were sporadic. Important geographic variations were observed in the frequencies of these populations, accompanied by year-to-year populational shifts. Epidemiology and histopathology studies showed the presence of the pathogens on vegetative organs year-round, particularly on olive leaves and branches, and on weeds. These represent inoculum reservoirs where secondary conidiation occurs, and conidia are then dispersed by spring rains reaching flowers and young fruits or by autumn rains reaching pre-mature fruits. Unripe fruits were colonized without showing symptoms up to penetration of the cuticle, but further colonization and symptom production was completed only as fruits matured. These findings challenge current control practices, particularly the timing of fungicide treatment, and contribute to improved disease management.
- Epidemiology, histopathology and aetiology of olive anthracnose caused by Colletotrichum acutatum and C. gloesporioides in PortugalPublication . Talhinhas, P.; Mota-Capitão, C.; Martins, S.; Ramos, Ana Paula; Neves-Martins, J.; Guerra-Guimarães, L.; Várzea, V.; Silva, M.C.; Sreenivasaprasad, S.; Oliveira, HelenaAnthracnose is an important disease affecting mature olive fruits, causing significant yield losses, and poor fruit and oil quality. In Portugal, high anthracnose incidence was recorded during 2003–2007 with 41%of 908 orchards surveyed displaying disease symptoms. In another 14% of the orchards, the pathogen was recorded in symptomless plants. Disease severity was on average 36%, frequently reaching 100%. In Portugal, anthracnose is endemic to neglected orchards of susceptible cultivars, but under favourable conditions it can also severely affect less susceptible cultivars. Pathogens were genetically heterogeneous, with Colletotrichum acutatum genetic group A2 as the most frequent (80%), followed by group A4 (12%) and group A5 along with C. gloeosporioides (3–4%), while groups A3 and A6 of C. acutatum were sporadic. Important geographic variations were observed in the frequencies of these populations, accompanied by year-to-year populational shifts. Epidemiology and histopathology studies showed the presence of the pathogens on vegetative organs year-round, particularly on olive leaves and branches, and on weeds. These represent inoculum reservoirs where secondary conidiation occurs, and conidia are then dispersed by spring rains reaching flowers and young fruits or by autumn rains reaching pre-mature fruits. Unripe fruits were colonized without showing symptoms up to penetration of the cuticle, but further colonization and symptom production was completed only as fruits matured. These findings challenge current control practices, particularly the timing of fungicide treatment, and contribute to improved disease management