Estacion Experimental La Mayora

Málaga, Spain

Estacion Experimental La Mayora

Málaga, Spain
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Heredia-Guerrero J.A.,University of Seville | Dominguez E.,Estacion Experimental La Mayora | Luna M.,Institute Microelectronica Of Madrid Imm Csic | Benitez J.J.,University of Seville | Heredia A.,University of Malaga
Chemistry and Physics of Lipids | Year: 2010

In the present work, we report the physico-chemical properties and structural characteristics of special polyhydroxy fatty acid nanoparticles after their fusion by means of attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), X-ray diffraction (XRD), differential scanning calorimetry (DSC), scanning electronic microscopy (SEM), atomic force microscopy (AFM), and light microscopy. All the characteristics and properties investigated have an important degree of similarity to the native plant cutin, the main biopolymer present in the plant cuticles. The supramolecular organization of these polymerized prime nanoparticles after their interaction on cellulose substrate and isolated cuticle samples, simulating the in vivo conditions in epidermal plant cells, strongly suggests a growth of these nanoparticles after a self-assembly process. © 2010 Elsevier Ireland Ltd. All rights reserved.


Pereira-Carvalho R.C.,National Center for Vegetable Crops Research | Pereira-Carvalho R.C.,University of Brasilia | Pereira-Carvalho R.C.,CSIC - Institute of Refrigeration | Boiteux L.S.,University of Brasilia | And 6 more authors.
Plant Disease | Year: 2010

The Ty-1 locus confers tolerance to monopartite and bipartite Begomovirus spp. (genus Begomovirus, family Geminiviridae) and this phenotype is improved in homozygous tomato lines. However, the gene Mi (Meloidogyne spp. resistance) is in repulsion phase linkage with Ty-1, which hampers the large-scale development of multiresistant inbred lines. Seventy-one Solanum (section Lycopersicon) accessions were whitefly inoculated with the bipartite Begomovirus sp. Tomato rugose mosaic virus (ToRMV) and simultaneously infested with a mixture of Meloidogyne incognita and M. javanica under greenhouse conditions in Brazil. Accessions were then transplanted into a nematode-infested field with natural ToRMV infection. A severity index was used to evaluate ToRMV reaction. Nematode evaluation was done by counting the number of galls per root system. Seventeen accessions with Meloidogyne spp. and ToRMV resistance were selected and evaluated in Spain against three monopartite Begomovirus spp. associated with the tomato yellow leaf curl virus disease, using infectious clones. Systemic infection was monitored by DNA hybridization. Five S. peruvianum accessions (PI-306811, PI-365951, LA-1609, LA-2553, and CNPH-1194) displayed nematode and broad-spectrum resistance to all Begomovirus spp. tested in both continents. From the breeding standpoint, accessions combining resistance to Meloidogyne spp. and to bipartite and monopartite Begomovirus spp. would be useful for the development of elite lines expressing all traits in homozygous condition. Copyright © 2010 The American Phytopathological Society.


Martin C.,Estacion Experimental La Mayora | Hormaza J.I.,Estacion Experimental La Mayora | Herrero M.,CSIC - Aula Dei Experimental Station
Acta Horticulturae | Year: 2010

The recovery and conservation of traditional fruit tree cultivars is a necessity that faces a short window of opportunity due to accelerating cultivar erosion. Several efforts to preserve old material took place before the widespread use of molecular markers, but often that material has not been conserved in ex situ germplasm collections. Here we present a case in Spain where a thorough inventory of local fruit tree species was performed in the 1950s with detailed data of the origin of each genotype. However, from most of that material the only remnants are an old stone collection. We developed a protocol in apricot (Prunus armeniaca) to obtain DNA from maternal tissues of the stones of a sufficient quality to be amplified by PCR. Then we compared the results obtained with the profiles obtained from cultivars currently conserved in ex situ germplasm collections. The results highlight the lost and preserved variability and help to prioritize the recovery of particular cultivars. The approach used in this work provides information on genetic erosion that has taken place in fruit tree species, but could also be applied to other plant species.


Garcia-Pedrajas M.D.,Estacion Experimental La Mayora
Methods in molecular biology (Clifton, N.J.) | Year: 2010

Gene deletion is one of the most powerful tools to study gene function. In the genomics era there is great demand for fast, simple high-throughput methods for gene deletion to study the roles of the large numbers of genes that are being identified. Here we present an approach that speeds up the process of generation of deletion mutants by greatly simplifying the production of gene deletion constructs. With this purpose we have developed a method, which we named DelsGate (Deletion via Gateway), that combines PCR and Gateway cloning technology together with the use of the I-SceI homing endonuclease to generate precise deletion constructs in a very simple, universal and robust manner in just 2 days. DelsGate consists of standard PCR of only the 5' and 3' 1 kb gene flanks directly followed by in vitro Gateway cloning and final generation of the circular deletion construct by in vivo recombination in Escherichia coli. For use in DelsGate we have modified a Gateway cloning vector to include selectable markers for the transformation of Ascomycetes and the Basidiomycete fungus Ustilago maydis. The PCR and transformation steps of DelsGate should be well suited for high-throughput approaches to gene deletion construction in fungal species. We describe here the entire process, from the generation of the deletion construct with DelsGate to the analysis of the fungal transformants to test for gene replacement, with the Basidiomycete fungus Ustilago maydis. Application of DelsGate to other fungal species is also underway. Additionally, we describe how this basic approach can be adapted to other genetic manipulations with minor changes. We specifically describe its application to create unmarked deletions in Ralstonia solanacearum, a Gram-negative phytopathogenic bacterium.


Garcia-Pedrajas M.D.,University of Georgia | Baeza-Montanez L.,Estacion Experimental La Mayora | Gold S.E.,University of Georgia
Molecular Plant-Microbe Interactions | Year: 2010

In Ustilago maydis, the causal agent of corn smut, the morphological transition from yeast to filamentous growth is inextricably linked to pathogenicity; budding haploid cells are saprobic and, upon mating of compatible strains, the fungus converts to dikaryotic filamentous growth and obligate parasitism. The filamentous dikaryon proliferates in the host plant, inducing tumor formation and undergoing additional morphological changes that eventually result in the production of melanized diploid teliospores. In an attempt to identify new trans -acting factors that regulate morphogenesis in U. maydis, we searched for the presence of common binding sequences in the promoter region of a set of 37 genes downregulated in the filamentous form. Putative cis-acting regulatory sequences fitting the consensus binding site for the Aspergillus nidulans transcription factor StuA were identified in 13 of these genes. StuA is a member of the APSES transcription factors which contain a highly conserved DNA-binding domain with a basic helix-loop-helix (bHLH)-like structure. This class of proteins comprises critical regulators of developmental processes in ascomycete fungi such as dimorphic growth, mating, and sporulation but has not been studied in any fungus of the phylum Basidiomycota. A search for StuA orthologs in the U. maydis genome identified a single closely related protein that we designated Ustl. Deletion of ust1 in budding haploid wild-type and solopathogenic strains led to filamentous growth and abolished mating, gall induction, and, consequently, in planta teliosporogenesis. Furthermore, cultures of ust1 null mutants produced abundant thick-walled, highly pigmented cells resembling teliospores which are normally produced only in planta. We showed that ssp1, a gene highly induced in teliospores produced in the host, is also abundantly expressed in cultures of ust1 null mutants containing these pigmented cells. Our results are consistent with a major role for ust1 in regulating dimorphism, virulence, and the sporulation program in U. maydis. © 2010 The American Phytopathological Society.


Martinez-Medina A.,CSIC - Experimental Station of El Zaidín | Fernandez I.,CSIC - Experimental Station of El Zaidín | Sanchez-Guzman M.J.,Estacion Experimental La Mayora | Jung S.C.,CSIC - Experimental Station of El Zaidín | And 2 more authors.
Frontiers in Plant Science | Year: 2013

Root colonization by selected Trichoderma isolates can activate in the plant a systemic defense response that is effective against a broad-spectrum of plant pathogens. Diverse plant hormones play pivotal roles in the regulation of the defense signaling network that leads to the induction of systemic resistance triggered by beneficial organisms [induced systemic resistance (ISR)]. Among them, jasmonic acid (JA) and ethylene (ET) signaling pathways are generally essential for ISR. However, Trichoderma ISR (TISR) is believed to involve a wider variety of signaling routes, interconnected in a complex network of cross-communicating hormone pathways. Using tomato as a model, an integrative analysis of the main mechanisms involved in the systemic resistance induced by Trichoderma harzianum against the necrotrophic leaf pathogen Botrytis cinerea was performed. Root colonization by T. harzianum rendered the leaves more resistant to B. cinerea independently of major effects on plant nutrition. The analysis of disease development in shoots of tomato mutant lines impaired in the synthesis of the key defense-related hormones JA, ET, salicylic acid (SA), and abscisic acid (ABA), and the peptide prosystemin (PS) evidenced the requirement of intact JA, SA, and ABA signaling pathways for a functional TISR. Expression analysis of several hormone-related marker genes point to the role of priming for enhanced JA-dependent defense responses upon pathogen infection. Together, our results indicate that although TISR induced in tomato against necrotrophs is mainly based on boosted JA-dependent responses, the pathways regulated by the plant hormones SA- and ABA are also required for successful TISR development. © 2013 Martínez-Medina, Fernández, Sánchez-Guzmán, Jung, Pascual and Pozo.


PubMed | Estacion Experimental La Mayora
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2010

Gene deletion is one of the most powerful tools to study gene function. In the genomics era there is great demand for fast, simple high-throughput methods for gene deletion to study the roles of the large numbers of genes that are being identified. Here we present an approach that speeds up the process of generation of deletion mutants by greatly simplifying the production of gene deletion constructs. With this purpose we have developed a method, which we named DelsGate (Deletion via Gateway), that combines PCR and Gateway cloning technology together with the use of the I-SceI homing endonuclease to generate precise deletion constructs in a very simple, universal and robust manner in just 2 days. DelsGate consists of standard PCR of only the 5 and 3 1 kb gene flanks directly followed by in vitro Gateway cloning and final generation of the circular deletion construct by in vivo recombination in Escherichia coli. For use in DelsGate we have modified a Gateway cloning vector to include selectable markers for the transformation of Ascomycetes and the Basidiomycete fungus Ustilago maydis. The PCR and transformation steps of DelsGate should be well suited for high-throughput approaches to gene deletion construction in fungal species. We describe here the entire process, from the generation of the deletion construct with DelsGate to the analysis of the fungal transformants to test for gene replacement, with the Basidiomycete fungus Ustilago maydis. Application of DelsGate to other fungal species is also underway. Additionally, we describe how this basic approach can be adapted to other genetic manipulations with minor changes. We specifically describe its application to create unmarked deletions in Ralstonia solanacearum, a Gram-negative phytopathogenic bacterium.


Rocha K.C.G.,São Paulo State University | Marubayashi J.M.,São Paulo State University | Navas-Castillo J.,Estacion Experimental La Mayora | Pavan M.A.,São Paulo State University | Krause-Sakate R.,São Paulo State University
Summa Phytopathologica | Year: 2010

From January/2007 to July/2008 a survey was carried out to evaluate the occurrence of begomoviruses in pepper and tomato crops from São Paulo State. Total DNA was extracted from 710 pepper and 103 tomato samples, and the presence of begomoviruses was tested by Polymerase chain reaction (PCR). The same samples were tested by Rolling Circle Amplification (RCA) followed by PCR, and some positive samples analyzed by RCA-RFLP and cleaved by the restriction enzyme HpaII to evaluate the genetic variability of these isolates. By PCR, 99 (13.94%) samples collected from pepper and 39 (37.86%) from tomato were positives for the presence of begomovirus, while by RCA-PCR 333 (46.90%) and 82 (79.61%) from pepper and tomato, respectively, indicating higher sensitivity of this technique. The 5' region of the coat protein (CP) gene and a segment of the intergenic region was analyzed indicating the presence of Tomato severe rugose virus (ToSRV) in pepper and tomato plants. However, the partial sequencing of clones from RCA products from a tomato sample indicated mixed infection of ToSRV with Tomato yellow vein streak virus (ToYVSV). By RCA-RFLP four restriction profiles were observed for ToSRV in pepper, while 18 profiles for begomovirus from tomato plants, indicating higher degree of genetic variability for begomovirus found in tomato plants compared to that in pepper plants.


PubMed | Estacion Experimental La Mayora
Type: Journal Article | Journal: Physiologia plantarum | Year: 2012

The tos1 (tomato osmotically sensitive) mutant, isolated from an in vitro screen of root growth during osmotic stress, was less sensitive to exogenous ABA, but accumulated more ABA under osmotic stress than WT plants. We assessed growth and water relations characteristics of hydroponically grown tos1 seedlings (in the absence of osmotic stress) at low and high evaporative demands. Growth of tos1 was severely inhibited at both high and low evaporative demands. Twenty DAS, WT and tos1 genotypes had a similar leaf water and turgor potential, but mature tos1 plants (45 day old) showed a significant diurnal loss of leaf turgor, with recovery overnight. Increased evaporative demand increased turgor loss of tos1 plants. High evaporative demand at the beginning of the day decreased stomatal conductance of tos1, without diurnal recovery, thus whole plant transpiration was decreased. De-topped tos1 seedlings showed decreased root hydraulic conductance and had a 1.4-fold increase in root ABA concentration. Impaired root function of tos1 plants failed to meet transpirational water demand and resulted in shoot turgor loss, stomatal closure and growth inhibition.


PubMed | Estacion Experimental La Mayora
Type: Journal Article | Journal: Plant science : an international journal of experimental plant biology | Year: 2011

Plant biomechanics combines the principles of physics, chemistry and engineering to answer questions about plant growth, development and interaction with the environment. The epidermal-growth-control theory, postulated in 1867 and verified in 2007, states that epidermal cells determine the rate of organ elongation since they are under tension, while inner tissues are under compression. The lipid cuticle layer is deposited on the surface of outer epidermal cell walls and modifies the chemical and mechanical nature of these cell walls. Thus, the plant cuticle plays a key role in plant interaction with the environment and in controlling organ expansion. Rheological analyses indicate that the cuticle is a mostly viscoelastic and strain-hardening material that stiffens the comparatively more elastic epidermal cell walls. Cuticle stiffness can be attributed to polysaccharides and flavonoids present in the cuticle whereas a cutin matrix is mainly responsible for its extensibility. Environmental conditions such as temperature and relative humidity have a plasticizing effect on the mechanical properties of cuticle since they lower cuticle stiffness and strength. The external appearance of agricultural commodities, especially fruits, is of great economic value. Mechanical properties of the cuticle can have a positive or negative effect on disorders like fruit cracking, fungal pathogen penetration and pest infestation. Cuticle rheology has significant variability within a species and thus can be subjected to selection in order to breed cultivars resistant to pests, infestation and disorders.

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