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- Alternative oxidase gene family in Hypericum perforatum L.: characterization and expression at the post-germinative phasePublication . Velada, Isabel; Cardoso, Hélia G.; Ragonezi, Carla; Nogales, Amaia; Ferreira, Alexandre; Valadas, Vera; Arnholdt-Schmitt, BirgitAlternative oxidase (AOX) protein is located in the inner mitochondrial membrane and is encoded in the nuclear genome being involved in plant response upon a diversity of environmental stresses and also in normal plant growth and development. Here we report the characterization of the AOX gene family of Hypericum perforatum L. Two AOX genes were identified, both with a structure of four exons (HpAOX1, acc. KU674355 and HpAOX2, acc. KU674356). High variability was found at the N-terminal region of the protein coincident with the high variability identified at the mitochondrial transit peptide. In silico analysis of regulatory elements located at intronic regions identified putative sequences coding for miRNA precursors and trace elements of a transposon. Simple sequence repeats were also identified. Additionally, the mRNA levels for the HpAOX1 and HpAOX2, along with the ones for the HpGAPA (glyceraldehyde-3-phosphate dehydrogenase A subunit) and the HpCAT1 (catalase 1), were evaluated during the post-germinative development. Gene expression analysis was performed by RT-qPCR with accurate data normalization, pointing out HpHYP1 (chamba phenolic oxidative coupling protein 1) and HpH2A (histone 2A) as the most suitable reference genes (RGs) according to GeNorm algorithm. The HpAOX2 transcript demonstrated larger stability during the process with a slight down-regulation in its expression. Contrarily, HpAOX1 and HpGAPA (the corresponding protein is homolog to the chloroplast isoform involved in the photosynthetic carbon assimilation in other plant species) transcripts showed a marked increase, with a similar expression pattern between them, during the post-germinative development. On the other hand, the HpCAT1 (the corresponding protein is homolog to the major H2O2-scavenging enzyme in other plant species) transcripts showed an opposite behavior with a down-regulation during the process. In summary, our findings, although preliminary, highlight the importance to investigate in more detail the participation of AOX genes during the post-germinative development in H. perforatum, in order to explore their functional role in optimizing photosynthesis and in the control of reactive oxygen species (ROS) levels during the process.
- Review of sewage sludge as a soil amendment in relation to current international guidelines: a heavy metal perspectivePublication . Nunes, Nuno; Ragonezi, Carla; Gouveia, Carla S. S.; Carvalho, Miguel Â. A. Pinheiro deOverexploitation of resources makes the reutilization of waste a focal topic of modern society, and the question of the kind of wastes that can be used is continuously raised. Sewage sludge (SS) is derived from the wastewater treatment plants, considered important underused biomass, and can be used as a biofertilizer when properly stabilized due to the high content of inorganic matter, nitrate, and phosphorus. However, a wide range of pollutants can be present in these biosolids, limiting or prohibiting their use as biofertilizer, depending on the type and origin of industrial waste and household products. Long-term applications of these biosolids could substantially increase the concentration of contaminants, causing detrimental effects on the environment and induce hyper accumulation or phytotoxicity in the produced crops. In this work, some critical parameters for soils and SS agronomic use, such as organic matter, nitrogen, phosphorous, and potassium (NPK), and heavy metals concentration have been reviewed. Several cases of food crop production and the accumulation of heavy metals after SS application are also discussed. SS production, usage, and legis lation in EU are assessed to determine the possibility of sustainable management of this bioresource. Additionally, the World Health Organization (WHO) and Food and Agriculture Organization (FAO) guidelines are addressed. The opportunity to produce bioenergy crops, employing sewage sludge to enhance degraded land, is also considered, due to energy security. Although there are numerous advantages of sewage sludge, proper screening for heavy metals in all the variants (biosolids, soil, food products) is a must. SS application requires appropriate strict guidelines with appropriate regulatory oversight to control contamination of agricultural soils.
- Contributo para o conhecimento da agrodiversidade no concelho de Santa Cruz, MadeiraPublication . Carvalho, Miguel A. A. Pinheiro de; Ragonezi, Carla; Macedo, Fabrício Lopes de; Antunes, Gonçalo; Freitas, Gregório; Nóbrega, HumbertoO presente trabalho procura analisar as alterações sofridas pela agrodiversidade no território do Santa Cruz. Este concelho localizado na costa sul da Madeira tem 81,5 km2, quatro patamares bioclimáticos e numerosas unidades agroecológicas (UAE). A evolução da agrodiversidade iniciou-se, com o povoamento do Arquipélago, descoberto inabitado em 1418. A agrodiversidade biofísica, específica e intraespecífica aumentou e diversificou-se, devido à construção de agrossistemas, introdução de culturas, sucessão de ciclos agrícolas, adaptação às condições agroecológicas, aparecimento de pragas e doenças e pela ação do homem. Os registos históricos indicam a introdução de um elevado número de culturas, bem como a utilização de 72 espécies agrícolas, no concelho. E, indicam como fundamentais no povoamento e economia as culturas do trigo e as cerealíferas (cevada, centeio), cana-sacarina, vinha e sumagre. Várias outras culturas desempenharam um papel importante na economia do concelho, sendo cultivadas para fins alimentares, comerciais, industriais, culturais ou religiosos. A adaptação destas espécies aos agrossistemas e às condições agroecológicas deu origem a um número indeterminado de variedades locais. Apesar das limitações da documentação disponível, foram encontradas referências a variedades locais cuja cultura remonta há 140 a 500 anos, nomeadamente o Trigo da Serra, castas Malvasia e Tinta Antiga de Gaula, pêra Caniça, Banana da terra, feijão Corno de carneiro e Couve de João Ferino, que podem ser relacionadas com o historial do concelho. Porém, desde meados do século XX, a agrodiversidade sofreu uma drástica redução com a diminuição das áreas cultivadas, o abandono de várias espécies agrícolas e a extinção de variedades locais. A análise desta agrodiversidade foi realizada, a fim de apurar as alterações históricas e geográficas ocorridas na agrodiversidade em onze culturas representativas do concelho, visando a sustentabilidade agronómica.
- Prediction of the Potential Distribution of Drosophila suzukii on Madeira Island Using the Maximum Entropy ModelingPublication . Macedo, Fabrício Lopes; Ragonezi, Carla; Reis, Fábio; Freitas, José G. R. de; Lopes, David Horta; Aguiar, António Miguel Franquinho; Cravo, Délia; Carvalho, Miguel A. A. Pinheiro deDrosophila suzukii is one of the main pests that attack soft-skinned fruits and cause significant economic damage worldwide. Madeira Island (Portugal) is already affected by this pest. The present work aimed to investigate the potential distribution of D. suzukii on Madeira Island to better understand the limits of its geographical distribution on the island using the Maximum Entropy modeling (MaxEnt). The resultant model provided by MaxEnt was rated as regular discrimination with the area under the curve (AUC, 0.7–0.8). Upon scrutinizing the environmental variables with the greatest impact on the distribution of D. suzukii, altitude emerged as the dominant contributor, with the highest percentage (71.2%). Additionally, elevations ranging from 0 to 500 m were identified as appropriate for the species distribution. With the results of the model, it becomes possible to understand/predict which locations will be most suitable for the establishment of the analyzed pest and could be further applied not only for D. suzukii but also for other species that hold the potential for substantial economic losses in this insular region.
- Molecular approach to characterize ectomycorrhizae fungi from Mediterranean pine stands in PortugalPublication . Ragonezi, Carla; Caldeira, A. Teresa; Martins, M. Rosário; Salvador, Cátia; Santos-Silva, Celeste; Ganhão, Elsa; Klimaszewska, Krystyna; Zavattieri, Maria AmelyStone pine (Pinus pinea L.), like other conifers, forms ectomycorrhizas (ECM), which have benefi cial impact on plant growth in natural environments and forest ecosystems. An in vitro co-culture of stone pine microshoots with pure mycelia of isolated ECM sporocarps was used to overcome the root growth cessation not only in vitro but also to improve root development during acclimation phase. Pisolithus arhizus (Scop.) Rauschert and Lactarius deliciosus (L. ex Fr.) S.F. Gray fungi, were col lected, pure cultured and used in in vitro co-culture with stone pine microshoots. Samples of P. arhizus and L. deliciosus for the in vitro co-cultures were collected from the pine stands southwest Portugal. The in situ characterization was based on their morphotypes. To confirm the identity of the collected material, ITS amplification was applied using the pure cultures derived from the sporo carps. Additionally, a molecular profile using PCR based genomic fingerprinting comparison was executed with other genera of Basidiomycetes and Ascomycetes. Our results showed the effective ness of the techniques used to amplify DNA polymorphic sequences, which enhances the characte rization of the genetic profile of ECM fungi and also provides an option to verify the fungus identity at any stage of plant mycorrhization.
- Histological studies of mycorrhized roots and mycorrhizal-like-structures in pine rootsPublication . Ragonezi, Carla; Zavattieri, MariaSeveral studies have shown the potential of using Ectomycorrhizal (ECM) fungi in conifer micropropagation to overcome the cessation of adventitious root development. In vitro inoculation promotes the re-growth of the root system induced previously by auxin treatments, facilitating acclimation and diminishing the losses of plants because of a weak root system that is incapable of water and nutrient absorption. During a series of mycorrhization experiments, cryostat and ultrafine cuts were used to study the morpho-histological transformation of the symbiotic roots. To obtain cryostat cuts from pine roots a method frequently used for animal tissue was adopted. Molecular methods allowed fungi identification in all the mycorrhization phases and in the acclimation of derived plants. Mycorrhizal-like-structures derived from in vitro culture and axenic liquid cultures of roots were microscopically analyzed and compare with mycorrhizal roots.
- Structure and floristic composition associated with an endangered species Beta patula Aiton (Amaranthaceae) in the Islands of Madeira ArchipelagoPublication . Nóbrega, Humberto; Freitas, Gregório; Zavattieri, M. A.; Ragonezi, Carla; Carvalho, Miguel Â. A. Pinheiro deTwenty-two native Crop Wild Relatives (CWR) occur in specific dry environments of Madeira Archipelago, like Desembarcadouro islet in Ponta de São Lourenço and Chão islet in Desertas Islands. Nine of them share the same gene pool with crop species included in Annex I of the International Treaty on Plant Genetic Resources for Food and Agriculture. Amongst them, Beta patula Aiton, an IUCN Critically Endangered species, has been studied in detail for in situ and ex situ conservation. The present paper summarises the information recorded during the Beta patula population assessment. Valuable information on plant communities associated with this species was obtained.
- Viral diagnosis in cultivars of Ipomoea batatas (L.) LamPublication . Andrade, Carlos G.; Silva, Emanuel M. da; Ragonezi, Carla; Carvalho, Miguel Â. A. Pinheiro deIpomoea batatas (L.) Lam. commonly known as sweet potato, is an important staple food worldwide, mainly due to its high nutritional value and yield. However, vegetative reproduction of sweet potato makes it more susceptible to viral infections, which threatens its productivity, quality, and difficult long-term preservation in germplasm banks. Also, it can act as a virus reservoir infecting the rest of the plant accessions in the bank collections. Hence, this work aimed to screen Begomovirus, Potyvirus, and Carlavirus infections in 16 traditional sweet potato cultivars from the germplasm collection of the ISOPlexis Germplasm Bank, Madeira, Portugal. The infection prevalence by these viruses among cultivars was 81.25%, 25.00%, and 6.25%, respectively; being ISOP1011 the only accession coinfected by Potyvirus and Carlavirus. The accessions ISOP1006, ISOP1010, and ISOP1047 were also coinfected by Begomovirus and Potyvirus, highlighting their vulnerability to viral infections. The ISOP1005 and ISOP1027 accessions were the only ones not infected by any of these viruses. The analysis of the partial sequence obtained from the Carlavirus detected in the accession ISOP1011, revealed the existence of an ORF that encodes for 93 amino acids of the catalytic domain of an RNA-directed RNA polymerase related to the Tymovirus protein family, as could be confirmed by comparison with proteins stored in UniProtKB. Multiple sequence alignment with these proteins showed that Motifs A and B of the catalytic domain were conserved. The search for sequence similarity with sequences deposited in GenBank reported a high sequence identity with Sweet potato yellow mottle virus (SPYMV) and Sweet potato chlorotic fleck virus (SPCFV). However, the 9-11% discrepancy in nucleotide sequence identity and a phylogenetic analysis carried out using the maximum probability method suggests the virus isolated from ISOP1011 is a new divergent strain of the SPCFV species.
- Different seaweeds use for iodine deficiency overcomePublication . Ferraz, Sónia; Ragonezi, Carla; Nunes, Nuno; Valente, Sofia; Carvalho, Miguel A. A. Pinheiro deIodine deficiency is one of the three most common nutritional deficiencies worldwide and is a global public health problem that affects particularly the young children, pregnant women and elderly. The consequences of iodine deficiency disorder (IDD) include goiter and cretinism. The traditional consumption of iodine via supplemented salt should be monitored to reduce population sodium intake due to hypertension problems and the uncertain bioavailability of iodine in the supplemented salt. One approach for an effective and cost-efficient iodine supplementation is the use of seaweeds in food intake. Seaweeds, among all foods, are the most famous and reliable source of natural iodine. In this minireview we pretend to explore the different uses of seaweeds to fill the iodine deficiency and improve life quality.
- Zoneamento Agroclimático da cultura da videira para a Ilha da Madeira - PortugalPublication . Macedo, Fabrício Lopes; Ragonezi, Carla; Carvalho, Miguel Ângelo Almeida Pinheiro deA cultura da vinha possuiu uma elevada importância na produção de vinhos de qualidade na Ilha da Madeira. No entanto para que a produção da cultura venha a produzir de maneira mais satisfatória é extremamente necessário o conhecimento dos locais mais propícios para o desenvolvimento da cultura. O objetivo do presente trabalho foi realizar um Zoneamento Agroclimático para a cultura da videira na Ilha da Madeira, visando fornecer dados para a tomada de decisão de produtores e técnicos na seleção de novos locais para implementação da cultura. Para tanto, foram utilizados dados de 11 estações meteorológicas convencionais localizadas em toda a Ilha da Madeira com períodos de observação de 1961 a 1990. A obtenção do deficit hídrico foi obtida por meio do balanço hídrico proposto por Thornthwaite & Mather. Para o cultivo da videira na Ilha da Madeira, todo litoral Sul da Ilha, assim como boa parte do Norte, são áreas propicias para o desenvolvimento da cultura; no entanto no litoral sul, irrigação complementar é necessária para satisfazer as necessidades hídricas da cultura. Já no litoral Norte é necessário ter alguns cuidados com possível aparecimento de doenças fúngicas, devido aos grandes volumes de precipitação presentes na área.
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