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- Profiling of lipophilic and phenolic phytochemicals of four cultivars from cherimoya (Annona cherimola Mill.)Publication . Santos, Sónia A. O.; Vilela, Carla; Camacho, João F.; Cordeiro, Nereida; Gouveia, Manuela; Freire, Carmen S. R.; Silvestre, Armando J.D.The lipophilic and phenolic extractives of the ripe mesocarp of four cherimoya cultivars ('Perry Vidal', 'Mateus I', 'Mateus III' and 'Funchal') from Madeira Island, were studied for the first time. The predominant lipophilic compounds are kaurene diterpenes (42.2-59.6%), fatty acids (18.0-35.6%) and sterols (9.6-23.7%). Kaur-16-en-19-oic acid is the major lipophilic component of all cultivars accounting between 554 and 1350mgkg(-1) of dry material. The studied fruits also contain a high variety of flavan-3-ols, including galloylated and non-galloylated compounds. Five phenolic compounds were identified for the first time: catechin, (epi)catechin-(epi)gallocatechin, (epi)gallocatechin, (epi)afzelechin-(epi)catechin and procyanidin tetramer. 'Mateus I' and 'Mateus III' cultivars present the highest content of phenolic compounds (6299 and 9603mgkg(-1) of dry weight, respectively). These results support the use of this fruit as a rich source of health-promoting components, with the capacity to prevent or delay the progress of oxidative-stress related disorders.
- Infection-Related Activation of the cg12 Promoter Is Conserved between Actinorhizal and Legume-Rhizobia Root Nodule SymbiosisPublication . Svistoonoff, Sergio; Laplaze, Laurent; Liang, Jingsi; Ribeiro, Ana; Gouveia, Manuela C.; Auguy, Florence; Fevereiro, Pedro; Franche, Claudine; Bogusz, DidierTwo nitrogen-fixing root nodule symbioses between soil bacteria and higher plants have been described: the symbiosis between legume and rhizobia and actinorhizal symbioses between plants belonging to eight angiosperm families and the actinomycete Frankia. We have recently shown that the subtilisin-like Ser protease gene cg12 (isolated from the actinorhizal plant Casuarina glauca) is specifically expressed during plant cell infection by Frankia. Here we report on the study of C. glauca cg12 promoter activity in the transgenic legume Medicago truncatula. We found that cg12 promoter activation is associated with plant cell infection by Sinorhizobium meliloti. Furthermore, applications of purified Nod factors and mycorrhizal inoculation failed to trigger expression of the cg12-reporter gene construct. This indicates that at least part of the transcriptional environment in plant cells infected by endosymbiotic nitrogen-fixing bacteria is conserved between legume and actinorhizal plants. These results are discussed in view of recent data concerning molecular phylogeny that suggest a common evolutionary origin of all plants entering nitrogen-fixing root nodule symbioses.
- Molecular cloning and characterization of cDNA encoding a Translocon-Associated Protein (TRAPδ) from the root-lesion nematode Pratylenchus goodeyiPublication . Pestana, Margarida; Abrantes, Isabel M. de O.; Gouveia, ManuelaThe translocon-associated protein (TRAP) complex comprises four subunits (α, β, γ, δ) and is located in the endoplasmic reticulum membrane at translocation sites. The TRAP complex is required for the efficient translocation of substrates and to correct or eliminate misfolded proteins. In this study, we described the cloning and characterization of a cDNA encoding a TRAP from the phytoparasitic nematode Pratylenchus goodeyi (Pg). The full-length cDNA had an estimated size of 690 bp and encodes a 177 amino acid peptide. The deduced protein after sequence analysis codes for TRAPδ subunit homologous to TRAPδ from other nematodes. The Pg-TRAPδ had a signal peptide indi cating a possible involvement in the transport and binding of other proteins at the endoplasmic reticulum membrane. The increase in relative expression of Pg trapδ, assessed by semi-quantitative PCR, was induced over time in nematodes exposed to a nematostatic/ nematicide extract of Solanum nigrum, suggesting that this gene product might be influenced by response mechanisms to stress in P. goodeyi. This is the first report of the cloning and characterization of trap cDNA from plant endoparasitic nematodes.
- Population decline in the critically endangered Musschia isambertoi (Campanulaceae) endemic to Desertas Islands (Madeira Archipelago) calls for urgent conservation managementPublication . Sequeira, M. Menezes de; Jardim, R.; Gouveia, M.; Góis-Marques, C. A.; Eddie, W. M. M.Macaronesian islands display a high degree of plant endemicity and correspond to some of the most threatened biodiversity hotspots due to several human driven impacts such as habitat loss, invasive species, overexploitation or climate change. The conservation status of the largest of the two known population of Musschia isambertoi M. Seq., R. Jardim, Magda Silva and L. Carvalho (Campanulaceae) was assessed recently. Field work was done in June 2018 in “Porto das Moças” Deserta Grande (Madeira Archipelago, Portugal), the locus typicus, where in 2006 close to 30 plants were detected with a high variation of age/size between individuals comprising both fully flowering and non-flowering adult plants as well as seedlings, and thus corresponding to a healthy population. This study assesses population size, age and plant size, and genetic, as well as floristic diversity. The total area of the site corresponds to approximately 1500 square meters. Ten individuals were detected, all corresponding to young seedlings not exceeding 15 cm high, and exhibiting approximately the same size and numbers of leaves (4–8). No adult plants or remains of adult plants were seen. Ecological data and direct herbivory evidence suggest that perennial plants are subjected to elimination during the short summer by the feral goat population. Mo lecular data based on ISSR markers show that the nine sampled individuals (out of ten individuals found) are genetically identical, possibly resulting from one parental plant. Results clearly suggest that Musschia isambertoi is very close to extinction. Urgent conservation measures are imperative and should include the immediate fencing of the population followed by the elimination of feral goats from Deserta Grande. The elimination of feral goats was initiated in 1996 (LIFE95 NAT/P/000125, 383,467.00 €) but stopped due to inappropriate conser vation policies of the former Services of the Natural Park of Madeira.
- Characterization of four defense-related genes up-regulated in root nodules of Casuarina glaucaPublication . Santos, Patrícia; Fortunato, Ana; Graça, Inês; Martins, Sandra Marina; Gouveia, Maria Manuela; Auguy, Florence; Bogusz, Didier; Ricardo, Cândido Pereira Pinto; Pawlowski, Katharina; Ribeiro, AnaActinorhizal plants are capable of high rates of nitrogen fixation, due to their capacity to establish a root nodule symbiosis with N2-fixing actinomycetes of the genus Frankia. Nodulation is an ontogenic process which requires a sequence of highly coordinated events. One of these mechanisms is the induction of defense-related events, whose precise role during nodulation is largely unknown. In order to contribute to the clarification of the involvement of defense-related genes during actinorhizal root-nodule symbiosis, we have analysed the differential expression of several genes with putative defense-related functions in Casuarina glauca nodules versus non inoculated roots. Four genes encoding a chitinase (CgChi1), a glutathione S-transferase (CgGst), a hairpin-inducible protein (CgHin1) and a peroxidase (CgPox4) were found to be up-regulated in mature nodules compared to roots. In order to find out to which extend were the encoded proteins involved in nodule protection, development or both, gene regulation studies in response to SA and wounding as well as phylogenetic analysis of the protein sequences were performed. These were further characterized through expression studies after SA-treatment and wounding, and by phylogenetic analysis. We suggest that CgChi1 and CgGst are involved in defense or microsymbiont control and CgPox4 is involved in nodule development. For CgHin1 the question “defense, development or both” remains open.
- Extinctions of introduced game species on oceanic islands: curse for hunters or conservation opportunities?Publication . Rocha, Ricardo; Sequeira, Miguel M.; Douglas, Leo R.; Gouveia, Manuela; Jardim, Roberto; Jesus, José; Jones, Holly P.; Russo, Danilo
- Efeitos de Solanum sisymbriifolium e S. nigrum sobre o nemátode-das-lesões-radiculares, Pratylenchus goodeyi, parasita da bananeiraPublication . Pestana, Margarida; Gouveia, Manuela; Abrantes, IsabelO objectivo deste trabalho foi desenvolver uma estratégia de luta contra o nemátode das-lesões-radiculares, Pratylenchus goo deyi, utilizando Solanum sisymbriifolium e S. nigrum como adubo verde ou biofumi gante. Inicialmente, foram realizados ensaios de patogenicidade, tendo-se verificado que apesar de P. goodeyi se ter reproduzido nas duas plantas, os factores de reprodução fo ram muito baixos (0,001), podendo ser con sideradas como resistentes ou, pelo menos, hospedeiros fracos. A incorporação de qual quer uma das espécies de Solanum no solo, com bananeiras infectadas com P. goodeyi, influenciou o crescimento das bananeiras e a reprodução de P. goodeyi foi superior nas ba naneiras apenas infectadas com o nemátode. Os extractos aquosos de S. sisymbriifolium foram os mais eficazes na mortalidade de P. goodeyi na concentração de 250 mg/ml. De acordo com os resultados obtidos, pode-se concluir que as plantas de S. sisymbriifolium e S. nigrum poderão ser utilizadas como adu bo verde e como biofumigante.
- In vitro evaluation of nematicidal properties of Solanum sisymbriifolium and S. nigrum extracts on Pratylenchus goodeyiPublication . Pestana, Margarida; Rodrigues, Mónica; Teixeira, Lucília; Abrantes, Isabel de O.; Gouveia, Manuela; Cordeiro, NereidaThe root-lesion nematode, Pratylenchus goodeyi, is among the most economically damaging parasites of banana plants. Nematode control can benefit from searching for novel bio-nematicides. The present study was carried out to assess the potential nematicidal properties of two Solanum species (Solanum sisynibriífolíum and S. nigrum) against P. goodeyi, using dichloromethane, acetone, ethanol and either cold or hot water extracts of the plants. Water extracts of both plants at a concentration of 10 mg ml" ' greatly affected nematode movement and also caused mortality. The analysis of sequential extracts at the same experimental concentrations showed that, although water extracts affect nematode mobility and mortality, the acetone extract from S. nigrum was the most efficient, causing 100% mortality after 23 h exposure. The results showed that S. sísymbríífolíum and S. nigrum extracts contain chemical components that induce morphological changes in the body structure of the root-lesion nematode, affect mobihty and cause mortality. The nematostatic and nematicidal potential of the extracts described herein merit further studies to find novel bio-nematicides against the root-lesion nematode.
- Heat shock-induced susceptibility of green coffee leaves and berries to Colletotrichum gloeosporioides and its association to PR and hsp70 gene expressionPublication . Chen, Z. J.; Ribeiro, A.; Silva, M. C.; Santos, P.; Guerra-Guimarães, L.; Gouveia, M.; Fernandez, D.; Rodrigues, C. J.Colletotrichum kahawae Bridge & Waller and C. gloeosporioides Penz are both coffee (Coffea arabica L.) pathogens but only the former induces damages on green coffee berries causing 70–80% losses, being known as coffee berry disease or CBD. The latter infects only very ripe coffee berries without destroying the seed but is also responsible for ‘dieback’ of branches in overbearing seasons. Detached green coffee berries and leaves inoculated with C. kahawae show usually symptoms 4–5 days after inoculation. With C. gloeosporioides penetration does not even occur. However, when green berries and leaves were subject to a previous heat shock of 55 8C for 30 s and then inoculated, there was a total breakdown of resistance regarding C. gloeosporioides and an acceleration of the infection process regarding C. kahawae. Heat shock induced susceptibility decreased with time for both fungi and became null about 40–48 h after the treatment for C. gloeosporioides. The temporary loss of resistance was associated with a transient downregulation of some PR genes, namely cachi3-1, cachi4-1 and capox-1, and by a transient upregulation of cahsp70. All genes analysed under this study were expressed constitutively, two of them, cachi3-1 and cachi4- 1, being up-regulated in inoculated controls. It is suggested that the synergistic presence of these constitutive defenses may be one of the mechanisms used by the plant to control resistance to C. gloeosporioides. Moreover we hypothesise that the protein coded by cahsp70 could facilitate the penetration of the pathogen. To our knowledge, this is the first time that the expression of PRs and Hsp70 coding genes is associated to heat shock-induced susceptibility.
- Genetic diversity in Hemileia vastatrixbased on RAPD markersPublication . Gouveia, M. Manuela C.; Ribeiro, Ana; Várzea, Vítor M. P.; Rodrigues Jr., Carlos J.Random amplified polymorphic DNA (RAPD) was used to assess the genetic structure of Hemileia vastatrix populations. Forty-five rust iso lates with different virulence spectra and from dif ferent hosts and geographical regions were ana lyzed. Out of 45 bands, generated with three RAPD primers, 35 (78%) were polymorphic and scored as molecular markers. Cluster analysis exhibits unstruc tured variability of this pathogen with regard to physiological race, geographical origin or host. The genotypic diversity (H9) inferred from Shannon’s in dex was higher than gene diversity (Ht), suggesting that diversity is distributed among clonal lineages. Estimates of gene diversity in Africa and Asia pop ulations were higher in total (Ht) as compared to within population diversity (Hs). Genetic differen tiation was considerable among coffee rust isolates from Africa (Gst 5 0.865) and Asia (Gst 5 0.768) but not among isolates from South America (Gst 5 0.266). We concluded that genetic diversity in H. vastatrix was moderately low and that the genetic differentiation among populations shows that asex ual reproduction is likely to play an important role in the population biology of this fungus. This should be taken into account for the development of breed ing programs.
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