Browsing by Author "Neves, D."
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- Anti-Phytophthora cinnamomi activity of Phlomis purpurea plant and root extractsPublication . Neves, D.; Caetano, P.; Oliveira, J. V.; Maia, Cristiana; Sousa, N.; Salgado, M.; Dionisio, L.; Magan, N.; Cravador, A.Phlomis purpurea (Lamiaceae), found in Quercus suber and Quercus ilex ssp. rotundifolia forest habitats in southern Portugal, is a non-host for the oomycete Phytophthora cinnamomi, the main biotic factor involved in cork oak and holm oak decline in the Iberian Peninsula. The effect of P. purpurea crude ethanol root extract was evaluated in vitro on P. cinnamomi mycelial growth, sporangial production, zoospore release and germination as well as on chlamydospore production and viability. The protection of cork oak against infection by the pathogen was also evaluated in planta. At 10 mg ml-1, in vitro inhibition of the pathogen structures was 85-100 %. In addition, P. purpurea plants were shown to protect Q. suber and Q. ilex from P. cinnamomi infection and to reduce the inoculum potential in glasshouse trials, indicating the ability to reduce root infection by the pathogen. The results suggest that P. purpurea has the potential to reduce disease spread and that their root extracts could provide candidate substances for control of the important pathogen, P. cinnamomi. © 2013 KNPV.
- Evaluation of the protective effect of Phlomis purpurea against Phytophthora cinnamomi in Fagaceae and of root metabolites involvedPublication . Neves, D.; Cravador, A.Phytophthora cinnamomi Rands devastates natural ecosystems and crops around the world causing enormous economic losses. The “montado” ecosystem is threatened by this highly aggressive pathogen. Much concern involving fungicide use highlighted the need to develop new environmentally friendly means of control. In this work, Phlomis purpurea L., which was recently reported to have activity against P. cinnamomi and protect susceptible Quercus ilex and Q. suber from infection, was further studied. To determine the protective activity of P. purpurea on the infection of susceptible species a greenhouse assay was implemented showing that, when planted next to the host, it was able to significantly reduce the average severity of host’s root symptoms. A field assay was set on naturally infested plots to determine the protective effect of this herbaceous plant towards Q. suber. P. purpurea significantly increased the emergence and survival of Q. suber. In both assays P. purpurea planted alone was able to completely eliminate P. cinnamomi. In order to study the effectiveness of P. purpurea root extracts (PRE) to control Q. suber and Q. ilex root infection by this oomycete, in vivo assays were set up. PRE at 10 mg ml-1 significantly inhibited P. cinnamomi zoospore infection of both plants. Moreover, PRE was able to elicit a defence response on Q. suber roots. To identify the active principle, bioactivity guided isolation was performed after fractionation of PRE. Isolation, purification and structural characterization led to identification of a novel nortriterpene, phlomispurpentaolone, containing the anti-P. cinnamomi activity. Moreover, P. purpurea metabolites produced constitutively and upon challenge with P. cinnamomi were quantified using LC-MS showing that phlomispurpentaolone is a phytoanticipin. Another goal was to confirm the resistance of P. purpurea to P. cinnamomi by histological techniques. P. purpurea roots were shown to have a constitutive strengthened exodermis that can act, per se, as a physical barrier for the penetration of P. cinnamomi.
- Genetic diversity of two evergreen oaks (Quercus suber L. and Q (ilex) rotundifolia Lam.) in Portugal using AFLP markersPublication . Coelho, A. C.; Lima, M. B.; Neves, D.; Cravador, A.The genetic variability of cork oak (Quercus suber, L.) in Portugal was evaluated by AFLP using five primer combinations. Three hundred and thirteen trees from three geographically contrasting regions exhibited a high level of genetic variation. The genetic profile of each individual is composed of 291 loci, randomly positioned in the genome and consists of monomorphic and polymorphic fragments. Similarities and dissimilarities among the individuals were quantitatively evaluated by numerical taxonomy. The overall sample shows a proportion of AFLP polymorphic markers of 71%, denoting a high level of variability. Ninety percent of the polymorphic markers identified in cork oak genotypes are uniformly distributed throughout the cork oak populations of Algarve, Alentejo and Trás-os-Montes regions. The coefficients of genetic similarity vary from 0.61 to 0.88 implying that 60% of fragments found are common. A sample of 52 holm oak [Quercus ilex subsp. rotundifolia (Lam.)] trees from overlapping areas was also analysed by AFLP with the same five primer combinations. However the codification of markers together with those selected on cork oak profiles was feasible with only one primer combination due to an apparent much higher polymorphism. AFLP and numerical taxonomy analysis enabled to differentiate the taxa and showed that the level of similarity observed between the profiles of the individuals from holm oak species was lower than that observed in cork oak, implying that apparently the degree of polymorphism is higher in Q. ilex subsp. rotundifolia than that quantified in Q. suber. A Bayesian approach was used to assess Q. suber total genetic diversity (Ht = 0.2534, P < 0.001) of which 1.7% (Fst = 0.0172, P < 0.001) was assigned to differences among populations. Analysis of molecular variance (AMOVA) showed that most genetic variation is comprised within populations (96%) while 3.6% is among populations (Φst = 0.036, P < 0.001). Differences among populations within geographic regions account for 2.6% (Φsc = 0.026, P < 0.001) of the total variation and only 1.3% (Φct = 0.013, P = 0.007) is attributed to variation among regions denoting little differentiation of populations over a range of 700 km.
- Genetic diversity of two evergreen oaks [Quercus suber (L.) and Quercus ilex subsp rotundifolia (Lam.)] in Portugal using AFLP markersPublication . Coelho, Ana Cristina; Lima, M. B.; Neves, D.; Cravador, AlfredoThe genetic variability of cork oak (Quercus suber, L.) in Portugal was evaluated by AFLP using five primer combinations. Three hundred and thirteen trees from three geographically contrasting regions exhibited a high level of genetic variation. The genetic profile of each individual is composed of 291 loci, randomly positioned in the genome and consists of monomorphic and polymorphic fragments. Similarities and dissimilarities among the individuals were quantitatively evaluated by numerical taxonomy. The overall sample shows a proportion of AFLP polymorphic markers of 71%, denoting a high level of variability. Ninety percent of the polymorphic markers identified in cork oak genotypes are uniformly distributed throughout the cork oak populations of Algarve, Alentejo and Tras-os-Montes regions. The coefficients of genetic similarity vary from 0.61 to 0.88 implying that 60% of fragments found are common. A sample of 52 holm oak [Quercus ilex subsp. rotundifolia (Lam.)] trees from overlapping areas was also analysed by AFLP with the same five primer combinations. However the codification of markers together with those selected on cork oak profiles was feasible with only one primer combination due to an apparent much higher polymorphism. AFLP and numerical taxonomy analysis enabled to differentiate the taxa and showed that the level of similarity observed between the profiles of the individuals from holm oak species was lower than that observed in cork oak, implying that apparently the degree of polymorphism is higher in Q. ilex subsp. rotun-difolia than that quantified in Q. suber. A Bayesian approach was used to assess Q. suber total genetic diversity (Ht = 0.2534, P < 0.001) of which 1.7% (Fst = 0.0172, P < 0.001) was assigned to differences among populations. Analysis of molecular variance (AMOVA) showed that most genetic variation is comprised within populations (96%) while 3.6% is among populations (Phi st = 0.036, P < 0.001). Differences among populations within geographic regions account for 2.6% (Phi sc = 0.026, P < 0.001) of the total variation and only 1.3% (Phi ct = 0.013, P = 0.007) is attributed to variation among regions denoting little differentiation of populations over a range of 700 km.
- In vitro and in vivo quantification of elicitin expression in Phytophthora cinnamomiPublication . Horta, Marília; Sousa, Nelson; Coelho, A. C.; Neves, D.; Cravador, A.The differential expression of four Phytophthora cinnamomi elicitin genes was analysed by Real Time RT-PCR. In in vitro cultures, the a-cinnamomin gene showed the highest level of expression, the b-cinnamomin gene (b-cin) was the most inducible, and the HAE transcripts were in low abundance. Transcription of all the elicitins was active during the active growth of the pathogen when infecting cork oak (Quercus suber) roots, and as host colonization progressed, the level of b-cin expression fell, while that of a-cin rose. In an antisense transgenic strain, the silencing of b-cin also negatively affected the expression of other elicitin genes in the cluster. The reduced in planta growth of the b-cin knock-out is related to the altered pattern of elicitin gene expression, supporting the idea that one of the functions of elicitins is related, directly or indirectly, with pathogenesis.
- Involvement of a cinnamyl alcohol dehydrogenase of Quercus suber in the defence response to infection by Phytophthora cinnamomiPublication . Coelho, A. C.; Horta, Marília; Neves, D.; Cravador, A.A gene encoding a potential NADPH-dependent cinnamyl alcohol dehydrogenase (QsCAD1) (GenBank accession no: AY362455) was identified in Quercus suber (cork oak). Its complete cDNA sequence was obtained by RACE-PCR, starting from total RNA extracted from roots of seedlings of Q. suber, infected with Phytophthora cinnamomi, the causal agent of the decline and sudden death of Q. suber and Quercus ilex subsp. rotundifolia in the Iberian Peninsula. Sequence information to perform the RACE-PCR was acquired from a polymorphic fragment (C9), specifically identified by cDNA-AFLP, in leaves of epicormic shoots of a cork oak tree that suffered sudden death. RT-PCR and hybridization analysis showed that the QsCAD1 gene is up-regulated in root seedlings of Q. suber infected with P. cinnamomi. QsCAD1 has a high structural homology with VR-ERE (Vigna radiata), an enzyme that detoxifies eutypine (produced by Eutypa lata, the causal agent of eutypa dieback of grapevines), to eutypinol, and with QrCAD1 (Q. ilex subsp. rotundifolia), EgCAD1 (Eucalyptus gunnii), MdCAD1 (Malus x domestica). Taken together, these results suggest that these enzymes, and namely QsCAD1 belong to a new group of CAD potentially involved in deactivation of toxins produced by phytopathogens.
- Molecular genetic analysis of a cattle population to reconstitute the extinct Algarvia breedPublication . Ginja, Catarina; Penedo, Maria; Sobral, M.; Matos, José; Borges, Carla; Neves, D.; Rangel-Figueiredo, Teresa; Cravador, A.Abstract Background Decisions to initiate conservation programmes need to account for extant variability, diversity loss and cultural and economic aspects. Molecular markers were used to investigate if putative Algarvia animals could be identified for use as progenitors in a breeding programme to recover this nearly extinct breed. Methods 46 individuals phenotypically representative of Algarvia cattle were genotyped for 27 microsatellite loci and compared with 11 Portuguese autochthonous and three imported breeds. Genetic distances and factorial correspondence analyses (FCA) were performed to investigate the relationship among Algarvia and related breeds. Assignment tests were done to identify representative individuals of the breed. Y chromosome and mtDNA analyses were used to further characterize Algarvia animals. Gene- and allelic-based conservation analyses were used to determine breed contributions to overall genetic diversity. Results Genetic distance and FCA results confirmed the close relationship between Algarvia and southern Portuguese breeds. Assignment tests without breed information classified 17 Algarvia animals in this cluster with a high probability (q > 0.95). With breed information, 30 cows and three bulls were identified (q > 0.95) that could be used to reconstitute the Algarvia breed. Molecular and morphological results were concordant. These animals showed intermediate levels of genetic diversity (MNA = 6.0 ± 1.6, Rt = 5.7 ± 1.4, Ho = 0.63 ± 0.19 and He = 0.69 ± 0.10) relative to other Portuguese breeds. Evidence of inbreeding was also detected (Fis = 0.083, P < 0.001). The four Algarvia bulls had Y-haplotypes H6Y2 and H11Y2, common in Portuguese cattle. The mtDNA composition showed prevalence of T3 matrilines and presence of the African-derived T1a haplogroup. This analysis confirmed the genetic proximity of Algarvia and Garvonesa breeds (Fst = 0.028, P > 0.05). Algarvia cattle provide an intermediate contribution (CB = 6.18, CW = -0.06 and D1 = 0.50) to the overall gene diversity of Portuguese cattle. Algarvia and seven other autochthonous breeds made no contribution to the overall allelic diversity. Conclusions Molecular analyses complemented previous morphological findings to identify 33 animals that can be considered remnants of the Algarvia breed. Results of genetic diversity and conservation analyses provide objective information to establish a management program to reconstitute the Algarvia breed.
- Molecular genetic analysis of a cattle population to reconstitute the extinct Algarvia breedPublication . Cravador, A.; Ginja, Catarina; Penedo, Maria; Sobral, M.; Matos, José; Borges, Carla; Neves, D.; Rangel-Figueiredo, TeresaBackground: Decisions to initiate conservation programmes need to account for extant variability, diversity loss and cultural and economic aspects. Molecular markers were used to investigate if putative Algarvia animals could be identified for use as progenitors in a breeding programme to recover this nearly extinct breed. Methods: 46 individuals phenotypically representative of Algarvia cattle were genotyped for 27 microsatellite loci and compared with 11 Portuguese autochthonous and three imported breeds. Genetic distances and factorial correspondence analyses (FCA) were performed to investigate the relationship among Algarvia and related breeds. Assignment tests were done to identify representative individuals of the breed. Y chromosome and mtDNA analyses were used to further characterize Algarvia animals. Gene- and allelic-based conservation analyses were used to determine breed contributions to overall genetic diversity. Results: Genetic distance and FCA results confirmed the close relationship between Algarvia and southern Portuguese breeds. Assignment tests without breed information classified 17 Algarvia animals in this cluster with a high probability (q > 0.95). With breed information, 30 cows and three bulls were identified (q > 0.95) that could be used to reconstitute the Algarvia breed. Molecular and morphological results were concordant. These animals showed intermediate levels of genetic diversity (MNA = 6.0 ± 1.6, Rt = 5.7 ± 1.4, Ho = 0.63 ± 0.19 and He = 0.69 ± 0.10) relative to other Portuguese breeds. Evidence of inbreeding was also detected (Fis = 0.083, P < 0.001). The four Algarvia bulls had Y-haplotypes H6Y2 and H11Y2, common in Portuguese cattle. The mtDNA composition showed prevalence of T3 matrilines and presence of the African-derived T1a haplogroup. This analysis confirmed the genetic proximity of Algarvia and Garvonesa breeds (Fst = 0.028, P > 0.05). Algarvia cattle provide an intermediate contribution (CB = 6.18, CW = -0.06 and D1 = 0.50) to the overall gene diversity of Portuguese cattle. Algarvia and seven other autochthonous breeds made no contribution to the overall allelic diversity. Conclusions: Molecular analyses complemented previous morphological findings to identify 33 animals that can be considered remnants of the Algarvia breed. Results of genetic diversity and conservation analyses provide objective information to establish a management program to reconstitute the Algarvia breed.