Browsing by Author "Cravador, Alfredo"
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- Cloning, characterization, in vitro and in planta expression of a necrosis-inducing Phytophthora protein 1 gene npp1 from Phytophthora cinnamomiPublication . Martins, Ivone M.; Meirinho, Sofia; Costa, Rodrigo; Cravador, Alfredo; Choupina, AltinoThe soil-borne oomycete Phytophthora cinnamomi is a highly destructive Phytophthora species associated with the decline of forest. This pathogen secretes a novel class of necrosis-inducing proteins known as Nep1-like proteins (NLPs). In this work, we report the sequencing and molecular characterization of one of these proteins, more specifically the necrosis-inducing Phytophthora protein 1 (NPP1). The ORF of the npp1 gene (EMBL database AM403130) has 768 bp encoding a putative peptide of 256 amino acids with a molecular weight of approximately 25 kD. In order to understand its function, in vitro gene expression was studied during growth in different carbon sources (glucose, cellulose, and sawdust), and at different times of infection, in vivo by RT-qPCR. The highest expression of the npp1 gene occurred in glucose medium followed by sawdust. In vivo infection of Castanea sativa roots with P. cinnamomi revealed a decrease in npp1 expression from 12 to 24 h; at 36 h its expression increased suggesting the existence of a complex mechanism of defense/attack interaction between the pathogen and the host. Expression of recombinant npp1 gene was achieved in Pichia pastoris and assessed by SDS-PAGE analysis of the protein secreted into the culture supernatant, revealing the presence of the NPP1 protein.
- Diversity of Phytophthora species in Valdivian rainforests and association with severe dieback symptomsPublication . Jung, Thomas; Durán, Alvaro; Stowasser, Eugenio Sanfuentes von; Schena, Leonardo; Mosca, Saveria; Fajardo, Sebastian; González, Mariela; Navarro Ortega, Angella D.; Bakonyi, Jozsef; Seress, Diana; Michal Tomšovský; Cravador, Alfredo; Maia, Cristiana; Marilia Horta Jung; Horta Jung, Marília; Jung, Thomas; Cravador, AlfredoThe Valdivian rainforest, one of the global hotspots of biodiversity, is a temperate rainforest originating as a Tertiary relic from the supercontinent Gondwana. In November 2014, a survey of Phytophthora diversity was performed in 13 natural forest stands and 20 forest streams and rivers in two protected areas near Valdivia and in a temperate montane forest in the Concepción area. One planted stand each of the introduced tree species Castanea sativa and Fagus sylvatica were also included. Using baiting assays, eight described species and four previously unknown taxa of Phytophthora were isolated from 86% of the 50 rhizosphere soil samples from seven of the eight tree species sampled in 12 forest stands, and from 20 streams: P. chlamydospora, P. cinnamomi, P. kernoviae, P. lacustris, P. plurivora, P. pseudosyringae, P. ×cambivora, P. ×stagnum, P. valdiviana nom. prov. from Clade 2b, P. madida nom. prov. from Clade 8a, and P. chilensis nom. prov. and P. pseudokernoviae nom. prov. The latter two species are the closest relatives of P. kernoviae from Clade 10. Phytophthora pseudokernoviae nom. prov. was also isolated from necrotic leaves of Drimys winteri. From the Valdivia river, a swarm of three Clade 6 hybrids was recovered. Each hybrid isolate resulted from multiple reticulation events with P. thermophila as maternal and both P. amnicola and P. chlamydospora as paternal parents. In addition, three previously unknown and recently described Nothophytophthora species, N. caduca, N. chlamydospora and N. valdiviana, were isolated from several forest streams. Phytophthora cinnamomi, the most common and widespread species in soils of native forests, was associated with severe dieback of Valdivian rainforest trees, in particular D. winteri, Luma apiculata, Nothofagus dombeyi and the endangered Saxegothaea conspicua. A first pathogenicity test demonstrated high aggressiveness of P. cinnamomi to several native tree species, including N. dombeyi, Blepharocalyx cruckshanksii and Gevuina avellana.
- Eight new Halophytophthora species from marine and brackish-water ecosystems in Portugal and an updated phylogeny for the genusPublication . Maia, Cristiana; Jung, M. Horta; Carella, G.; Milenković, I.; Janoušek, J.; Tomšovský, M.; Mosca, S.; Schena, L.; Cravador, Alfredo; Moricca, S.; Jung, T.During an oomycete survey in December 2015, 10 previously unknown Halophytophthora taxa were isolated from marine and brackish water of tidal ponds and channels in saltmarshes, lagoon ecosystems and river estuaries at seven sites along the Algarve coast in the South of Portugal. Phylogenetic analyses of LSU and ITS datasets, comprising all described Halophytophthora species, the 10 new Halophytophthora taxa and all relevant and distinctive sequences available from GenBank, provided an updated phylogeny of the genus Halophytophthora s.str. showing for the first time a structure of 10 clades designated as Clades 1-10. Nine of the 10 new Halophytophthora taxa resided in Clade 6 together with H. polymorphica and H. vesicula. Based on differences in morphology and temperature-growth relations and a multigene (LSU, ITS, Btub, hsp90, rpl10, tigA, cox1, nadh1, rps10) phylo-geny, eight new Halophytophthora taxa from Portugal are described here as H. brevisporangia, H. cele-ris, H. frigida, H. lateralis, H. lusitanica, H. macrosporangia, H. sinuata and H. thermoambigua. Three species, H. frigida, H. macrosporangia and H. sinuata, have a homothallic breeding system while the remaining five species are sterile. Pathogenicity and litter decomposition tests are underway to clarify their pathological and ecological role in the marine and brackish-water ecosystems. More oomycete surveys in yet undersurveyed regions of the world and population genetic or phylogenomic analyses of global populations are needed to clarify the origin of the new Halophytophthora species.
- 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.
- High diversity of Phytophthora species in natural ecosystems and nurseries of Portugal: detrimental side effect of plant introductions from the age of discovery to modern globalizationPublication . Horta Jung, Marília; Maia, Cristiana; Mora-Sala, Beatriz; Abad-Campos, Paloma; Schena, Leonardo; Mosca, Saveria; Carella, Giuseppe; Moricca, Salvatore; Nechwatal, Jan; Dionísio, Lídia; Cravador, Alfredo; Jung, ThomasBetween 2010 and 2015, an extensive survey of Phytophthora diversity was performed across Portugal. Baiting and direct isolation tests obtained 48 Phytophthora taxa in 62 of 70 forest stands (89%) from 148 of 302 rhizosphere soil samples (49%) and 25 of 29 tree species (86%), and from bark cankers of Alnus lusitanica and Fagus sylvatica ; from 33 of 34 rivers and forest streams (97%); and in 12 of 13 forest nurseries (92%) from 54 of 87 root ball samples (61%) and 16 of 21 tree species (76%). The 799 Phytophthora isolates belonged to 28 known and three recently described species; one designated and two new non‐hybrid taxa; and five designated and nine new hybrid taxa. The majority, 34 Phytophthora taxa (71%), are introduced aliens in Europe while nine (19%) and five (10%) taxa are of cryptic and native origin, respectively. Phytophthora cinnamomi was the most common species in forests (56%) and forest nurseries (61%). Multiple first records for Portugal and partly Europe of both Phytophthora taxa and tree declines and new host– Phytophthora associations were found. Isolation rates from forest soil differed considerably between declining (65%) and healthy trees (10%), regardless of the tree species. The widespread infestations of nurseries with aggressive wide‐host range pathogens like P . cinnamomi , P . pseudocryptogea , P . × cambivora , P . multivora and P . plurivora , and host‐specific pathogens like P . alticola , P . quercetorum and P . quercina , will inevitably cause their further spread to the wider environment, exacerbating pathogen threats to forest ecosystems and thus weakening their resilience to climatic extremes.
- A metabolome analysis and the immunity of Phlomis purpurea against Phytophthora cinnamomiPublication . Neves, Dina; Figueiredo, Andreia; Maia, Marisa; Laczko, Endre; Pais, Maria Salomé; Cravador, AlfredoPhlomis purpurea grows spontaneously in the southern Iberian Peninsula, namely in cork oak (Quercus suber) forests. In a previous transcriptome analysis, we reported on its immunity against Phytophthora cinnamomi. However, little is known about the involvement of secondary metabolites in the P. purpurea defense response. It is known, though, that root exudates are toxic to this pathogen. To understand the involvement of secondary metabolites in the defense of P. purpurea, a metabolome analysis was performed using the leaves and roots of plants challenged with the pathogen for over 72 h. The putatively identified compounds were constitutively produced. Alkaloids, fatty acids, flavonoids, glucosinolates, polyketides, prenol lipids, phenylpropanoids, sterols, and terpenoids were differentially produced in these leaves and roots along the experiment timescale. It must be emphasized that the constitutive production of taurine in leaves and its increase soon after challenging suggests its role in P. purpurea immunity against the stress imposed by the oomycete. The rapid increase in secondary metabolite production by this plant species accounts for a concerted action of multiple compounds and genes on the innate protection of Phlomis purpurea against Phytophthora cinnamomi. The combination of the metabolome with the transcriptome data previously disclosed confirms the mentioned innate immunity of this plant against a devastating pathogen. It suggests its potential as an antagonist in phytopathogens’ biological control. Its application in green forestry/agriculture is therefore possible.
- A metabolome analysis and the immunity of Phlomis purpurea against Phytophthora cinnamomiPublication . Neves, Dina; Figueiredo, Andreia; Maia, Marisa; Laczko, Endre; Pais, Maria Salomé; Cravador, AlfredoPhlomis purpurea grows spontaneously in the southern Iberian Peninsula, namely in cork oak (Quercus suber) forests. In a previous transcriptome analysis, we reported on its immunity against Phytophthora cinnamomi. However, little is known about the involvement of secondary metabolites in the P. purpurea defense response. It is known, though, that root exudates are toxic to this pathogen. To understand the involvement of secondary metabolites in the defense of P. purpurea, a metabolome analysis was performed using the leaves and roots of plants challenged with the pathogen for over 72 h. The putatively identified compounds were constitutively produced. Alkaloids, fatty acids, flavonoids, glucosinolates, polyketides, prenol lipids, phenylpropanoids, sterols, and terpenoids were differentially produced in these leaves and roots along the experiment timescale. It must be emphasized that the constitutive production of taurine in leaves and its increase soon after challenging suggests its role in P. purpurea immunity against the stress imposed by the oomycete. The rapid increase in secondary metabolite production by this plant species accounts for a concerted action of multiple compounds and genes on the innate protection of Phlomis purpurea against Phytophthora cinnamomi. The combination of the metabolome with the transcriptome data previously disclosed confirms the mentioned innate immunity of this plant against a devastating pathogen. It suggests its potential as an antagonist in phytopathogens’ biological control. Its application in green forestry/agriculture is therefore possible.
- Molecular cloning of two genes induced by alpha-interferon in human cells.Publication . Moutschen, S.; Wathelet, M.; Cravador, A.; Defilippi, P.; Huez, G.; Content, J.; Cravador, AlfredoIFN-induce proteins as well as IFN-induced enzymes have been considered for several years as possible mediators of the various interferon actions.
- Multiple new cryptic pathogenic Phytophthora species from Fagaceae forests in Austria, Italy and PortugalPublication . Jung, Thomas; Horta Jung, Marília; Cacciola, Santa Olga; Cech, Thomas; Bakonyi, Jozsef; Seress, Diana; Mosca, Saveria; Schena, Leonardo; Seddaiu, Salvatore; Pane, Antonella; Lio, Gaetano Magnano di San; Maia, Cristiana; Cravador, Alfredo; Franceschini, Antonio; Scanu, BrunoDuring surveys of Phytophthora diversity in natural and semi-natural Fagaceae forests in Austria, Italy and Portugal, four new cryptic species were isolated from rhizosphere soil samples. Multigene phylogeny based on nuclear ITS, beta-tubulin and HSP90 and mitochondrial cox1 and NADH1 gene sequences demonstrated that two species, P. tyrrhenica and P. vulcanica spp. nov., belong to phylogenetic Clade 7a, while the other two species, P. castanetorum and P. tubulina spp. nov., clustered together with P. quercina forming a new clade, named here as Clade 12. All four new species are homothallic and have low optimum and maximum temperatures for growth and very slow growth rates at their respective optimum temperature. They differed from each other and from related species by a unique combination of morphological characters, cardinal temperatures, and growth rates. Pathogenicity of all Phytophthora species to the root system of their respective host species was demonstrated in soil infestation trials.
- Structure, anti-Phytophthora and anti-tumor activities of a nortriterpenoid from the rhizome of Phlomis purpurea (Lamiaceae)Publication . Mateus, Maria Da Conceição; Neves, Dina; Dacunha, Bruno; Laczko, Endre; Maia, Cristiana; Teixeira, Rúben; Cravador, AlfredoTo investigate bioactive compounds potentially involved in the biotic interactions exhibited by Phlomis purpurea (Lamiaceae) in rhizospheres infested with Phytophthora cinnamomi, the plant rhizome was chemically analysed. The nortriterpenoid (175)-2 alpha,3 alpha,11 alpha,23,24-pentahydroxy-19(18 -> 17)-abeo-28-norolean-12-en-18-one, was isolated and its structure was elucidated by comprehensive spectroscopic analysis, chiefly using 2D NMR experiments, and X-ray analysis. It was shown to be exuded by roots and to exhibit anti-Phytophthora and antitumor activities. (C) 2016 Published by Elsevier Ltd.