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During 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. Portuguese Science and Technology Foundation (FCT); Portuguese national funds the European BiodivERsA project RESIPATH: Responses of European Forests and Society to Invasive Pathogens [BIODIVERSA/0002/2012]; Czech Ministry for Education, Youth and Sports; European Regional Development Fund [CZ.02.1.01/0.0/0.0/15_003/0000453]; Hungarian Scientific Research Fund (OTKA) [K101914]; European Union's Horizon research and innovation programme [635646]; POnTE (Pest Organisms Threatening Europe); Sardinian Regional Government info:eu-repo/semantics/publishedVersion

The 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., Project COMBATINTA/SP2.P11/02 Interreg IIIA—Cross-Border Cooperation Spain-Portugal, financed by The European Regional Development Fund and by national funding from the Portuguese Ministério da Ciência e do Ensino Superior (MCES) (PTDC/AGR-AAM/67628/2006)., info:eu-repo/semantics/publishedVersion

The strong association between Phytophthora cinnamomi and the mortality and decline of Quercus suber and Q. ilex subsp. rotundifolia has been known for two decades. The ability of elicitins secreted by this pathogen to trigger defence responses in these Quercus against itself was evaluated in this work. Biomass quantification by quantitative real-time PCR revealed a significant decrease in pathogen colonization of Q. suber roots after 24 h pre-treatment with α- and β-cinnamomin. In Q. suber and Q. ilex roots pre-treated with α-cinnamomin, hyphae were unable to reach and colonize the vascular cylinder and showed cytoplasmic disorganization in all the roots observed as contrasted with non-pre-treated roots. The pathogen was restricted to the intercellular spaces of the cortical parenchyma and the concomitant accumulation of electron dense materials was observed in contact with the hyphae. Furthermore, ROS (reactive oxygen species) production and the enzymatic activities of superoxide dismutase, catalase and peroxidase were compared in infected and non-infected Quercus roots in time course trials. There was a significant increase in the production of hydrogen peroxide (H2O2) and superoxide anion (O2 •-) and an enhanced activity of the enzymes in infected roots was observed at each time point. When comparing with elicitin non-treated roots, the α-cinnamomin-treated roots in interaction with P. cinnamomi showed a decrease in ROS accumulation and an increase of the enzyme activities. The overall results were consistent with an induction by the cinnamomins which initiated defence responses against the pathogen invasion of roots. Finally, elicitins were immunolocalized in the contact zone of P. cinnamomi hyphae with epidermal host cells, plasmalemma outer cytoplasm and around the intracellular hyphae in the vacuoles of invaded epidermal cells.

[EN] Background: Phlomis plants are a source of biological active substances with potential applications in the control of phytopathogens. Phlomis purpurea (Lamiaceae) is autochthonous of southern Iberian Peninsula and Morocco and was found to be resistant to Phytophthora cinnamomi. Phlomis purpurea has revealed antagonistic effect in the rhizosphere of Quercus suber and Q. ilex against P. cinnamomi. Phlomis purpurea roots produce bioactive compounds exhibiting antitumor and anti-Phytophthora activities with potential to protect susceptible plants. Although these important capacities of P. purpurea have been demonstrated, there is no transcriptomic or genomic information available in public databases that could bring insights on the genes underlying this anti-oomycete activity. Results: Using Illumina technology we obtained a de novo assembly of P. purpurea transcriptome and differential transcript abundance to identify putative defence related genes in challenged versus non-challenged plants. A total of 1,272,600,000 reads from 18 cDNA libraries were merged and assembled into 215,739 transcript contigs. BLASTX alignment to Nr NCBI database identified 124,386 unique annotated transcripts (57.7%) with significant hits. Functional annotation identified 83,550 out of 124,386 unique transcripts, which were mapped to 141 pathways. 39% of unigenes were assigned GO terms. Their functions cover biological processes, cellular component and molecular functions. Genes associated with response to stimuli, cellular and primary metabolic processes, catalytic and transporter functions were among those identified. Differential transcript abundance analysis using DESeq revealed significant differences among libraries depending on post-challenge times. Comparative cyto-histological studies of P. purpurea roots challenged with P. cinnamomi zoospores and controls revealed specific morphological features (exodermal strips and epi-cuticular layer), that may provide a constitutive efficient barrier against pathogen penetration. Genes involved in cutin biosynthesis and in exodermal Casparian strips formation were up-regulated. Conclusions: The de novo assembly of transcriptome using short reads for a non-model plant, P. purpurea, revealed many unique transcripts useful for further gene expression, biological function, genomics and functional genomics studies. The data presented suggest a combination of a constitutive resistance and an increased transcriptional response from P. purpurea when challenged with the pathogen. This knowledge opens new perspectives for the understanding of defence responses underlying pathogenic oomycete/plant interaction upon challenge with P. cinnamomi., This work was supported by the project PTDC/AGR-CFL/100217/2008 and the grant SFRH/BD/66016/2009, both funded by Fundacao para a Ciencia e Tecnologia (FCT). The APC charge was supported by the project UID/BIA/04325/2013 - MEDTBIO (FCT). The COST Office and the European Council provided financial support to the Short Term Scientific Mission (COST-STSM-FP0801-10084).

During various surveys of Phytophthora diversity in Europe, Chile and Vietnam slow growing oomycete isolates were obtained from rhizosphere soil samples and small streams in natural and planted forest stands. Phylogenetic analyses of sequences from the nuclear ITS, LSU, β-tubulin and HSP90 loci and the mitochondrial cox1 and NADH1 genes revealed they belong to six new species of a new genus, officially described here as Nothophytophthora gen. nov., which clustered as sister group to Phytophthora. Nothophytophthora species share numerous morphological characters with Phytophthora: persistent (all Nothophytophthora spp.) and caducous (N. caduca, N. chlamydospora, N. valdiviana, N. vietnamensis) sporangia with variable shapes, internal differentiation of zoospores and internal, nested and extended (N. caduca, N. chlamydospora) and external (all Nothophytophthora spp.) sporangial proliferation; smooth-walled oogonia with amphigynous (N. amphigynosa) and paragynous (N. amphigynosa, N. intricata, N. vietnamensis) attachment of the antheridia; chlamydospores (N. chlamydospora) and hyphal swellings. Main differing features of the new genus are the presence of a conspicuous, opaque plug inside the sporangiophore close to the base of most mature sporangia in all known Nothophytophthora species and intraspecific co-occurrence of caducity and non-papillate sporangia with internal nested and extended proliferation in several Nothophytophthora species. Comparisons of morphological structures of both genera allow hypotheses about the morphology and ecology of their common ancestor which are discussed. Production of caducous sporangia by N. caduca, N. chlamydospora and N. valdiviana from Valdivian rainforests and N. vietnamensis from a mountain forest in Vietnam suggests a partially aerial lifestyle as adaptation to these humid habitats. Presence of tree dieback in all forests from which Nothophytophthora spp. were recovered and partial sporangial caducity of several Nothophytophthora species indicate a pathogenic rather than a saprophytic lifestyle. Isolation tests from symptomatic plant tissues in these forests and pathogenicity tests are urgently required to clarify the lifestyle of the six Nothophytophthora species. info:eu-repo/semantics/publishedVersion

[EN] An analysis of incidence of Phytophthora spp. in 732 European nurseries producing forest transplants, larger specimen trees, landscape plants and ornamentals, plus 2525 areas in which trees and shrubs were planted, is presented based on work conducted by 38 research groups in 23 European countries between 1972 and 2013. Forty-nine Phytophthora taxa were recorded in 670 nurseries (91.5%); within these nurseries, 1614 of 1992 nursery stands (81.0%) were infested, although most affected plants appeared healthy. In forest and landscape plantings, 56 Phytophthora taxa were recovered from 1667 of 2525 tested sites (66.0%). Affected plants frequently showed symptoms such as crown thinning, chlorosis and dieback caused by extensive fine root losses and/or collar rot. Many well-known highly damaging host¿Phytophthora combinations were frequently detected but 297 and 407 new Phytophthora¿host associations were also observed in nurseries and plantings, respectively. On average, 1.3 Phytophthora species/taxa per infested nursery stand and planting site were isolated. At least 47 of the 68 Phytophthora species/taxa detected in nurseries and plantings were exotic species several of which are considered well established in both nurseries and plantings in Europe. Seven known Phytophthora species/taxa were found for the first time in Europe, while 10 taxa had not been previously recorded from nurseries or plantings; in addition, 5 taxa were first detections on woody plant species. Seven Phytophthora taxa were previously unknown to science. The reasons for these failures of plant biosecurity in Europe, implications for forest and semi-natural ecosystems and possible ways to improve biosecurity are discused., The authors are grateful to all public and private owners of nurseries and plantings who contributed to this extensive study. T. Jung acknowledges the support by the Regione Autonoma della Sardegna, Visiting Professor Program at the University of Sassari, Italy. The authors also thank the COST Office and the European Council for providing the European COST Actions FP0801 (http://www.cost.eu/domains_actions/fps/Actions/FP0801) and FP 1002 (http://www.cost.eu/domains_actions/fps/Actions/FP1002), the EU projects FOR-THREATS and ISEFOR, and BiodivERsA project RESIPATH as platforms for stimulating discussions on the nursery pathway and possible management solutions.

To 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.

The scope of this study was to identify sequence polymorphisms in the β -1,4-galactosyltransferase-I gene (B4GALT1), the gene which encodes the catalytic part of lactose synthase enzyme. Exons of the gene were scanned for the presence of allelic variation among a sample of 400 Iranian Holsteins, using the single strand conformation polymorphism technique followed by sequencing. Nine polymorphic nucleotide sites and 16 different genotypes of B4GALT1 were identified. We concluded therefore that the β-1,4-galactosyltransferase-I gene was polymorphic in Holsteins. Considering the biological roles of the B4GALT1 gene, we suggest that further studies are critical to investigate the biological effects of the SNPs reported in this study.

The 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 Concepcion 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.

The virulence of two wild type (PA45 and PA37) and two genetically modified (13C: hygromycin resistant; FATSS: hygromycin resistant and β-cin knock-down) Phytophthora cinnamomi strains towards cork oak (Quercus suber) was assessed via a quantitative evaluation of disease symptoms arising from a soil infestation assay, and by a histological analysis of root colonization. Comparison of virulence, as expressed by symptom severity, resulted in the following ranking: highly virulent (wild type strains), medium virulence (strain 13C) and weakly virulent (FATSS). Both transgenic strains were compromised in their virulence, as expressed by symptom severity, but strain 13C was much less affected than FATSS. Microscopic observation showed that the FATSS strain was unable to effectively invade the root, while 13C and the two wild type strains were all able to rapidly colonize the whole root, including the vascular tissue. These results strengthen the notion that elicitins are associated, either directly or indirectly, with the infection process of Phytophthora.

The morphology of a sample of four bulls and 43 cows, presumed to be descendants of the extinct cattle breed ‘Algarvia’ (AG), was used to assign their relationship with animals from other Portuguese autochthonous breeds – Arouquesa (AR), Barrosa˜ (BA), Cachena (CA), Marinhoa (MA), Maronesa (MO), Minhota (MN), Mirandesa (MI), (only bulls), Alentejana (AL), Garvonesa (GA), Mertolenga (ME) and Preta (PR). Standard numerical taxonomic methods were applied to a set of 183 (cows) and 170 (bulls) traits, to derive average pairwise taxonomic distances among the sample of 257 cows and 76 bulls. Distance coefficients (morphological index of distance) ranged from 0.22 to 2.62 (cows) and from 0.49 to 2.13 (bulls). Unweighted pair group method using arithmetic averages (UPGMA)-based phenograms and a principal coordinate analysis showed that bulls were highly clustered and cows showed a tendency to cluster according to their geographical and breed origin. The AG population grouped together with GA, AL, ME and MN breeds in the Red Convex group. The average taxonomic distance among breeds was 1.02, the highest being 1.39 (ME versus BA) and the lowest being 0.64 (MA versus AR). The approach allowed for the identification of a phenotypically differentiated set of animals, comprising 19 cows and four bulls representative of the AG breed, and which can be targeted in further studies aiming at the recovery of this extinct breed.

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-cinna- momin gene (b-cin) was the most inducible, and the HAE transcripts were in low abundance. Tran- scription 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 reduction 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. 2009 Published by Elsevier Ltd.

The genetic diversity of 15 carob ( Ceratonia siliqua L.) cultivars located in an experimental field from Algarve (Portugal) was evaluated over 7 years using 12 fruit and seed phenotypic characters, in order to characterize carob cultivars. The values of morphological traits obtained by cultiv ar were compared with those from other countries of the Mediterranean basin. Statistically significant differences were found between cultivars for al l characters which were examined, what indicates a high genetic diversity. The relationship among these characters was analyzed by principal component analysis (PCA) resulting in the separation of these cultivars classed in four groups (clusters I–IV) and in four ungrouped cultivars. A three dimension of the model was found to be significant and explained 74.5% of the total variation, in which the first component accounting for 34.6% of the total variation is dominated by fruit characters, while the second component is dominated by seed characters. Cultivars plotted on the left-lower quadrant on the space determined by principal components 1 and 2 are characterized by fruits with high seed yield more appropriated for industrial rentability. The correlation analyses established by cultivar provided a specific understanding about the way how fruit and seed characteristics correlate within each cultivar. This approach can be useful for the development of a breeding programme, aiming to increase the seed yield, seed thickness, individual and total seed weight by fruit, characteristics that are determinant to improve the industrial exploitation of carob.

cDNA-AFLP methodology was used to gain insight into gene fragments differentially present in the mRNA profiles of Quercus suber roots infected with zoospores of Phytophthora cinnamomi at different post challenge time points. Fifty-three transcript-derived fragments (TDFs) were identified and sequenced. Six candidate genes were selected based on their expression patterns and homology to genes known to play a role in defence. They encode a cinnamyl alcohol dehydrogenase2 (QsCAD2), a protein disulphide isomerase (QsPDI), a CC-NBS-LRR resistance protein (QsRPc), a thaumatin-like protein (QsTLP), a chitinase (QsCHI) and a 1,3-β-glucanase (QsGlu). Evaluation of the expression of these genes by quantitative polymerase chain reaction (qPCR) revealed that transcript levels of QsRPc, QsCHI, QsCAD2 and QsPDI increased during the first 24 h post-inoculation, while those of thaumatin-like protein decreased. No differential expression was observed for 1,3-β-glucanase (QsGlu). Four candidate reference genes, polymerase II (QsRPII), eukaryotic translation initiation factor 5A (QsEIF-5A), β-tubulin (QsTUB) and a medium subunit family protein of clathrin adaptor complexes (QsCACs) were assessed to determine the most stable internal references for qRT-PCR normalization in the Phytophthora-Q. suber pathosystem in root tissues. Those found to be more stable, QsRPII and QsCACs, were used as internal reference in the present work. Knowledge on the Quercus defence mechanisms against biotic stress is scarce. This study provides an insight into the gene profiling of a few important genes of Q. suber in response to P. cinnamomi infection contributing to the knowledge of the molecular interactions involving Quercus and root pathogens that can be useful in the future to understand the mechanisms underlying oak resistance to soil-borne oomycetes. Electronic supplementary material The online version of this article (doi:10.1186/2193-1801-3-613) contains supplementary material, which is available to authorized users.

Background: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. Results: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at www.corkoakdb.org. Conclusions: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response., This project was funded by "Fundacao para a Ciencia e a Tecnologia" (FCT) within a National Consortium (COEC - Cork Oak ESTs Consortium) that supported 12 sub-projects (SOBREIRO/033, 035, 014, 034, 015, 017, 038, 019, 029, 039, 030, 036/2009). The authors further wish to acknowledge FCT for ten doctoral (BD) and post doctoral (BPD) fellowships (Tania Almeida: SFRH/BD/44410/2008, Tiago Capote: SFRH/BD/69785/2010, Ines Chaves: SFRH/BPD/20833/2004, Ana S. Fortunato: SFRH/BPD/47563/2008, Marilia Horta: SFRH/BPD/63213/2009, Liliana Marum: "SFRH/BPD/47679/2008, Andreia Miguel: SFRH/BD/44474/2008, Margarida Rocheta: SFRH/BPD/64905/2009, Tatiana E. Santo: SFRH/BD/47450/2008, Monica Sebastiana: SFRH/BPD/25661/2005). Andreas Bohn, Nelson J.M. Saibo, Rita Teixeira were supported by the Programa Ciencia 2007, financed by POPH (QREN) and Isabel A. Abreu, Susana Araujo, Dora Batista, A. Margarida Fortes, Jorge A.P. Paiva, Sonia Goncalves by Programa Ciencia 2008, also funded by POPH (QREN). A Margarida Santos was funded through iBET (PEst-OE/EQB/LA0004/2011). Maintenance of the CorkOakDB is supported by the Instituto Gulbenkian de Ciencia.

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.

Oomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. They are able to secrete a glucanase inhibitor protein (GIP) that inhibits the activity of endoglucanases (EGases) involved in defense responses against infection. One of the most widely distributed and aggressive Phytophthora species, with more than 1,000 host plants is P. cinnamomi. In this work we report the sequencing and characterization of a class of GIPs secreted by Phytophthora cinnamomi. The gip gene from P. cinnamomi has a 937 bp ORF encoding a putative peptide of 312 deduced amino acids. The expression of this gene was studied during growth in different carbon sources (glucose, cellulose and sawdust), by RT-qPCR and its level of expression was evaluated at five time points. The highest expression of gip gene occurred in sawdust at 8 h of induction. In vivo infection of C. sativa revealed an increase in gip expression from 12 to 24 h. At 36 h its expression decreased suggesting that a compensatory mechanism must occur in plant.

The present preliminary study attempts to establish associations between milk production traits and genetic polymorphisms at the GH gene in the Algarvia goat. The DNA of 108 goats of the indigenous Portuguese Algarvia breed was evaluated. Single-strand conformation polymorphisms (SSCP) were identified at the five exons of the goat growth hormone (gGH) gene. Two conformational patterns were found in each of exons 1 and 2, four in exon 3, six in exon 4 and five in exon 5. An association between these SSCP patterns with milk, fat and protein production, and fat and protein content was examined. Patterns F/F of exon 4 and A/A of exon 5 were positively associated with milk production (P

Elicitins are a group of highly conserved proteins secreted by species of Phytophthora and a species of the related genus Pythium, Pythium vexans. Some of these proteins act as inducers of the necrotic hypersensitive-like response and the associated systemic acquired resistance phenomenon, in some species. We cloned and characterised the cinnamomin-beta and -alpha genes and two related elicitin genes from Phytophthora cinnamomi. These four open reading frames (ORFs) are clustered in tandem pairs. Two out of these four genes present homologies with the basic and acidic elicitin groups; but the two others encode, if expressed, elicitin isoforms exhibiting homologies with the class II of highly acidic elicitins.

In response to the need for a faster, more reliable method for identifying Phytophthora cinnamomi in cork oak soils in Portugal, a simple, fast, sensitive molecular identification method is described. It is based on a colorimetric assay which involves an oligonucleotide capture probe covalently immobilised on microtitration wells, a multi-biotinylated oligonucleotide detection probe and the PCR-amplified target DNA. The target DNA is a 349 bp DNA fragment partially covering the 3′-translated and 3′-untranslated regions of the cinnamomin gene. When the specificity of the PCR reaction was evaluated in vitro using isolates of P. cinnamomi and eight other Phytophthora species, including the related P. cambivora , it was specific to P. cinnamomi . When 30 isolates of P. cinnamomi from oak roots in southern Portugal were assayed, 26 gave a strong positive response. The assay has a sensitivity of about 2–5 genome equivalents of P. cinnamomi . The reason for the negative response of four isolates remains unclear.

The β(1,4)-galactosyltransferase-I gene (β4galt1) encodes the catalytic part of the enzyme lactose synthase, responsible of lactose synthesis in the mammary gland. The complete coding region of the gene was screened for the presence of allelic variation among a sample of 1,200 Iranian Holstein cows, using PCR-SSCP technique followed by sequencing. Nine polymorphic nucleotide sites were identified-one in exons I and VI, two in exons II and III, and three in exon V. Altogether 18 different genotypes were assigned. Statistical analysis showed that the genotypes of Β4GALT1 significantly affect milk, lactose, protein and total solid productions in both the first and second lactation (P < 0.001). Variance component analysis considering restricted maximum likelihood showed that the major factor making differences in milk, lactose, protein and total solid productions among the studied cow is the β4galt1 genotype. We concluded that the β4galt1 gene is potentially associated with milk production traits in dairy cows and should be considered for further studies on genetics of the milk production traits.

Several forest species are severely affected by Phytophthora cinnamomi. The contribution of this oomycete to forest decline and dieback has been broadly reported. In particular, it is consensual that it is the causal agent of ink disease in Castanea sativa. It has been associated with the severe decline of Quercus species, namely the Q. suber and Q. ilex dieback in Portugal and Spain, and has been responsible for the infection of numerous native species and crops. This pathogen persists in the soil or on plant material in the form of chlamydospores allowing the infection of living root tissues when environmental conditions are favorable.

The decline of cork oak (Quercus suber) stands in Iberian Peninsula is associated with infection by Phytophthora cinnamomi. Most Phytophthora species secrete elicitins, which can enhance defence reactions against some pathogens. Here cytological and physiological effects of the elicitins cryptogein and capsicein on cork oak root infection by P. cinnamomi were evaluated. The progression of the pathogen in root tissue and its effects on total fatty acid (TFA) composition of roots and leaves were analysed in seedlings. Net photosynthesis (P n ), stomatal conductance (g s ), chlorophyll a fluorescence measurements (quantum yield of linear electron transport ϕ e , photochemical quenching q P, non-photochemical quenching NPQ) and carotenoid determinations were carried out in well established (4 months) plants. In elicitin-treated roots, 2 days after inoculation, the pathogen which presented loss of viability and membrane degradation was mainly restricted to the intercellular spaces of the cortical parenchyma, and did not reach the vascular cylinder. Electron dense materials accumulated in the intercellular spaces of the cortex next to disorganized hyphae, suggested to be related with defence reactions. Cryptogein (or its interaction with P. cinnamomi) induced enhanced lipid synthesis in leaves, which may contribute to preserve membrane stability. P. cinnamomi decreased P n , g s , ϕ e , and q P, whereas elicitin-treated plants displayed values similar to controls. Overall, the results indicated a resistance response of cork oak against this oomycete, induced by the elicitins.

Glucose is the major precursor of lactose synthesis in the mammary gland. Lactose the major carbohydrate and osmolyte of milk, controls milk volume and its concentration. Glycosylated haemoglobin (HbG) is a retrospective measure of mean blood glucose level and it is largely unaffected by recent physiological conditions and environmental events. The purposes of this study were to determine the correlations between lactose traits and other milk production traits in dairy cattle and to investigate whether HbG level can be correlated with milk and lactose production traits. Here, HbG percentage, milk and lactose production traits including milk yield, lactose, protein, SNF, total solid and fat percentages and yields were measured in 485 second calved Iranian Holstein cattle. Statistically significant negative correlations were established between HbG and milk yield (r = -0.88), lactose yield (r = -0.83), SNF yield (r = -0.81), protein yield (r = -0.79) and total solid yield (r = -0.74). Positive correlations were established between lactose yield and milk (r = 0.96), protein (r = 0.81), SNF (r = 0.92) and total solid (r = 0.79) yields. The negative correlation between HbG and milk and total lactose production is probably related to the higher glucose demands in the lactating mammary gland of more productive cows. The positive correlation between lactose yield and milk, protein, SNF and total solid yield indicates that the level of lactose synthesis influences milk production traits in ways other than merely via its osmolytic action.

The oomycete P. cinnamomi has been described as strong contributing factor to the decline of cork oak and holm oak stands occurring in the Iberian Peninsula. There are no eradication methods available against this pathogen. Molecular mechanisms of oomycete — host interaction are not well understood, but it is known that oomycete species can induce biochemical and physiological processes in their host plants through a diverse array of virulence or avirulence molecules, known as effectors. Several Phytophtora effector molecules, the elicitins, are able to induce cellular defence responses in plants. Among these elicitors the 10- KDa (elicitins) are able to enhance plant defences in a systemic acquired resistance manner, against infection of different pathogens.

Phytophthora cinnamomi has been reported to be regularly associated with cork and holm oak decline. This oomycete secretes elicitins, a group of unique highly conserved proteins that can enhance plant defence reactions. In our previous work it was shown that the absorption of one of these elicitins─ α-CIN─ by the roots of cork and holm oak at concentrations of 500 μg and 1 mg/ml reduced P. cinnamomi viability and its progression into internal tissues. The objectives of the present work were to study the restriction of host tissue colonization and the loss of pathogen viability in the roots treated with lower concentration of α-CIN, and to verify structural alterations at cellular level induced by this elicitin in aerial part of the plantlets. Roots of Quercus suber seedlings two month old were immersed in aqueous α-CIN solutions [250 µg/ml] and [100 µg/ml] during 24 h. Roots were then inoculated with P. cinnamomi. The samples pre-treated with α-CIN at the two concentrations and inoculated with P. cinnamoni revealed similar histological and cytological aspects. The hyphae were mainly intercellularly located, restricted to the external cortex (Figs.A and B). The treatment induced loss of pathogen viability and it was evident the pathogen membranar degradation, loss of organelles and reduction of

Although carob (Ceratonia siliqua L.) is of great economic importance little is still known about the pattern of genetic variation within this species. Morphological characteristics based on 31 fruit and seeds of continuous characters determinant for agro-industrial uses, were compared with RAPD and AFLP markers for assessing genetic distances in 68 accessions of carob trees, from different cultivars, varieties and eco-geographic regions of Algarve. Eighteen selected RAPD primers applied to the 68 accessions produced a total of 235 fragments ranging from 200 to 2000 bp, of which 93 (40%) were polymorphic. Four AFLP selective primer combinations generated a total of 346 amplification fragments of which 110 were polymorphic. The average level of polymorphism based on four primer combinations was 31.8%. The phenetic trees based on RAPD and AFLP analyses gave high co-phenetic correlation values, and were found to be consistent in general with the analysis of morphological data, carried out on the same accessions. A number of RAPD and AFLP markers were found to be diagnostic for ‘Canela’ cultivar and 13 wild ungrafted trees.

Expression of POU1F1 gene, a member of the POU homeodomain family of transcription factors, is necessary for normal differentiation, development and survival of three anterior pituitary cell types (thyrotrophs, somatotrophs and lactotrophs) and for the proper expression of growth hormone (GH), prolactin (PRL), thyroid-stimulating hormone (TSH) genes and POU1F1 gene itself. Alternative splicing forms of this gene have been reported in different species, with few functional studies. Apart from the POU1F1-Wild-type with the expected length, in this work we isolated three additional splicing variants: POU1F1-beta, with a 78 bp insert in the trans-activation domain; POU1F1-gamma that lacks exon 3 and POU1F1-delta that lacks exons 3, 4 and 5. Four different protein isoforms were also detected by Western blot in the sheep pituitary tissue. Functional assays were performed to study the trans-activation of GH and PRL promoters by the splicing variants. Regarding the PRL promoter, the beta variant presented only 12% of the Wild-type trans-activation capacity. Variants gamma and delta showed no capacity to trans-activate PRL promoter. Both gamma and delta variants acted as repressors of Wt, reducing significantly the trans-activation made by Wt alone (p

POU1F1 (PIT-1/GHF-1) is a transcription factor with critical role in the transcriptional regulation of multiple genes in the pituitary and also important for the survival, differentiation and proliferation of three pituitary cell types. To understand the regulation of POU1F1 gene in Ovis aries we report its cloning, sequencing and characterization. The sequenced 5787 bp included six exons and two complete introns. Ovine POU1F1 gene has a high level of conservation with its bovine, human and rat counterparts showing 98.2%, 91.2% and 86.2% of similarity at the coding level, respectively. All six exons were analyzed for polymorphism detection in 100 animals of the Portuguese indigenous ovine breed 'Churra da Terra Quente'. One polymorphism was found at codon 58 in exon 2, in one allele of 4 animals leading to a change from cysteine to tyrosine (2% allelic frequency). In exon 3 two polymorphisms were detected: a G to A transition altering a glycine to an asparagine at codon 89 in one allele of one animal (0.5% allelic frequency) and another G to A transition at codon 105 converting an alanine into a threonine in one allele of 3 animals (1.5% allelic frequency). These polymorphisms might change the structure of the POU1F1 protein and modify gene-expression. In intron 4, an A to G transition was detected in one allele of six animals (3% allelic frequency). Exons 1, 4 and 6 showed no polymorphisms.

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. r 2007 Elsevier Ltd. All rights reserved.

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.

En Portugal, la enfermedad de la “seca” se ha descrito en los Quercus de hoja perenne (Quercus suber L. and Q.ilex subsp. rotundifolia Lam.) desde el final del siglo XIX. La mortalidad de estas especies afecta, particularmente las regiones centrales y meridionales del país, siendo uno de los problemas forestales más graves. Phytophthora cinnamomi Rands es el principal patógeno responsable de la mortalidad de alcornoques y encinas en Portugal. Se han desarrollado varios estudios teniendo como objetivo una mejor comprensión del efecto de la acción de P. cinnamomi en el decaimiento de los Quercus. El actual trabajo describe resultados preliminares de algunos de estos estudios.___________________________________In Portugal, the decline disease has been described in evergreen oaks (Quercus suber L. and Q.ilex subsp. rotundifolia Lam.) since the end of the 19th century. The mortality of these species affects, particularly the central and southern regions of the country, being one of the most severe forest problems. Phytophthora cinnamomi Rands is the main pathogen responsible for the cork and holm oak mortality in Portugal. Several studies have been developed aiming at a better understanding of the effect of the P. cinnamomi action on the cork oak trees decline. The present work describes preliminary results of some of these studies.

The crystal structure of the elicitin h-cinnamomin (h-CIN) was determined in complex with ergosterol at 1.1 Au resolution. h-CIN/ergosterol complex crystallized in the monoclinic space group P21, with unit cell parameters of a=31.0, b=62.8, c=50.0 Au and b=93.4- and two molecules in the asymmetric unit. Ligand extraction with chloroform followed by crystallographic analysis yielded a 1.35 Au structure of h-CIN (P43212 space group) where the characteristic elicitin fold was kept. After incubation with cholesterol, a new complex structure was obtained, showing that the protein retains, after the extraction procedure, its ability to complex sterols. The necrotic effect of h-CIN on tobacco was also shown to remain unchanged. Theoretical docking studies of the triterpene lupeol to h-CIN provided an explanation for the apparent inability of h-CIN to bind this ligand, as observed experimentally. D 2005 Elsevier B.V. All rights reserved.

Avec la domestication des animaux de rente, les éleveurs ont cherché d'une part, à accroître les performances de leurs animaux en sélectionnant dans la population mise à leur disposition les sujets les plus performants pour la (les) caractéristique(s) recherchée(s) et d'autre part, à réduire l'impact des zoonoses en sélectionnant les animaux résistants. Toutefois, si cette approche de la sélection a permis inconsciemment de favoriser au sein de la population les génotypes intéressants pour les critères recherchés, il importe de garder en mémoire que la production et la santé animale résultent non seulement du potentiel génétique mais également d'interactions entre l'alimentation, le statut physiologique, les facteurs environnementaux et le savoir-faire de l'éleveur.

The present study suggests the existence of an association between milk production traits and genetic polymorphisms at the growth hormone (GH) gene in the Portuguese indigenous Serrana goat. TheDNAfrom 229 animals of two ecotypes (Jarmelista and Ribatejano)was analysed by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique revealing a high degree of genetic polymorphism at the goat GH (gGH) gene. Two conformation patterns were detected in exons 1 and 2, 6 in exon 3, 10 in exon 4 and 5 in exon 5. The evaluation of an association effect between these SSCP patterns with milk, fat and protein yields and fat and protein percentages suggests a positive effect of pattern A/B of exon 4 for Ribatejano ecotype and of pattern A/B of exon 2 for Jarmelista ecotype with milk yield (P < 0.05) and of pattern A/B of exon 1 and pattern B/B of exon 2 with protein percentage (P < 0.05) for Ribatejano ecotype. The results support the hints suggested by previous studies of the importance of the gGH gene as a candidate gene for marker-assisted selection in goat breeds and suggest that exon 4 is a preferential target for further investigation on mutations that influence milk yield variation.

Phytophthora and Pythium species are among the most aggressive plant pathogens, as they invade many economically important crops and forest trees. They secrete large amounts of 10 kDa proteins called elicitins that can act as elicitors of plant defence mechanisms. These proteins may also induce a hypersensitive response (HR) including plant cell necrosis, with different levels of toxicity depending on their pI. Recent studies showed that elicitins function as sterol carrier proteins. The crystallographic structure of the highly necrotic recombinant beta-cinnamomin (beta-CIN) from Phytophthora cinnamomi has been determined at 1.8 Angstrom resolution using the molecular-replacement method. beta-CIN has the same overall structure as beta-cryptogein (beta-CRY), an elicitin secreted by Phytophthora cryptogea, although it shows a different surface electrostatic potential distribution. The protein was expressed in Pichia pastoris and crystallized in the triclinic space group with two monomers in the asymmetric unit. The interface formed by these two monomers resembles that from beta-CRY dimer, although with fewer interactions.

Evaluation of the genetic diversity for six genes in forty animals of the Portuguese indigenous sheep breed (Ovis aries) “Churra da Terra Quente” was done. A non-radioactive method to allow single-strand conformation polymorphism (SSCP) detection was optimised, starting from genomic DNAand PCR amplification of seven fragments: exon 1 of the a-lactalbumin gene; exons 10 and 11 of the as1-casein gene; exon 7 of the b-casein gene; exon 4 of the k-casein gene; exons 4 and 5 of the growth hormone gene and exon 6 of the growth hormone receptor gene. Polymorphisms were detected in five of the PCR products. Only k-casein and growth hormone receptor were monomorphic. a-lactalbumin and as1-casein exons showed three conformational patterns, b-casein and growth hormone exon 4 showed two electrophoretic patterns and growth hormone exon 5 showed five conformational patterns. These data provide evidence that “Churra da Terra Quente” has a high genetic variability, which opens interesting prospects for future selection programs and also for preservation strategies. Also, our data show that PCR-SSCP is an appropriate tool for evaluating genetic variability.

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A total of 195 bulls of eight Portuguese beef cattle breeds (Alentejana, Arouquesa, Barros , Maronesa, Marinhoa, Mertolenga, Mirandesa and Preta) were genotyped for the GH AluI polymorphism by the polymerase chain reaction and restriction length polymorphism (PCRRFLP). The genotype and gene frequencies for each breed were determined and shown to be quite variable among the breeds. The overall gene frequencies for L and V were 0.759 and 0.241, respectively. The relation between the bGH-AluI polymorphism and growth performances was ascertained in 168 of the animals analysed. According to our results there is a significant association between the genotypes LL and LV of the bGH and the average live body weight of the animals of the breeds Alentejana, Marinhoa and Preta.

Cinnamomin (CIN) belongs to a family of 10 kDa proteins designated as elicitins. Some of these proteins induce a hypersensitive response in diverse plant species, leading to resistance against fungal and bacterial plant pathogens. CIN was crystallized by the vapour-diffusion method using either ammonium sulfate or polyethyleneglycol (PEG) as precipitants in solutions buffered at around pH 7. These crystals are isomorphous and belong to the triclinic space group, with unit-cell parameters a = 31.69, b = 36.99, c = 44.09 Angstrom, alpha = 76.86, beta = 84.41, gamma = 80.26 degrees. A frozen crystal diffracted X-rays beyond 1.45 Angstrom resolution on a synchrotron-radiation source. info:eu-repo/semantics/publishedVersion

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