Primera noticia de la presencia de Opuntia robusta var. larreyi (Weber) en España

Recientemente han sido identificados en España, de forma provisional, ejemplares de la especie Opuntia robusta, variedad larreyi (Weber). Los ejemplares, ubicados en Madrid, están siendo estudiados por el Departamento de Producción Vegetal: Botánica y Protección Vegetal de la E.T.S.I. Agrónomos de Madrid con el objeto de evaluar su producción de biomasa.

Estudio sobre la influencia de la inoculación de las rizobacterias Azospirillum brasilense y Pseudomonas fluorescens sobre el desarrollo morfológico y el contenido de aceites esenciales en las especies aromáticas Ocimum basilicum var. genovesse, Ocimum basilicum var. minimum, Petroselinum sativum var. lisa y Salvia officinalis.

En el presente estudio se analiza la influencia de la inoculación con Azospirillum brasilense, con Pseudomonas fluorescens y la inoculación conjunta con ambas rizobacterias en las especies de plantas aromáticas Ocimum basilicum var. genovesse, Ocimum basilicum var. minimum, Petroselinum sativum var. lisa y Salvia officinalis. Se evaluará su desarrollo morfológico, atendiendo a tres parámetros: la longitud del tallo, el peso fresco y la superficie foliar. Así como el posible incremento en el contenido de aceite esencial que pueda tener la planta tratada. El cultivo se llevó a cabo en alveolos de ForestPot® 300, sobre mezcla de turba y vermiculita 3:1, con riego diario y sin adición de fertilizantes. Los resultados indican que en todas las especies y en todos los apartados estudiados, la inoculación de las rizobacterias produjo un incremento del desarrollo y del contenido de aceite esencial en comparación con el tratamiento Control, excepto en el caso de la longitud de las dos variedades de O. basilicum al inocularlas con P. fluorescens, en las que produjo una ligera disminución respecto al Control. En el caso de P. sativum var. lisa, solo las plantas que fueron inoculadas sobrevivieron. A partir de estos resultados, puede decirse que la inoculación con estas rizobacteras promotoras del crecimiento puede tener una gran importancia como sustitución de fertilizantes minerales, obteniéndose de este modo una producción más ecológica y respetuosa con el medio.

Coevolución virus-planta: impacto de las infecciones virales en poblaciones silvestres de Chiltepín (capsicum annuum var. Aviculare (dierbach) d’arcy & eshbaugh) en México.

El impacto negativo que tienen los virus en las plantas hace que estos puedan ejercer un papel ecológico como moduladores de la dinámica espacio-temporal de las poblaciones de sus huéspedes. Entender cuáles son los mecanismos genéticos y los factores ambientales que determinan tanto la epidemiología como la estructura genética de las poblaciones de virus puede resultar de gran ayuda para la comprensión del papel ecológico de las infecciones virales. Sin embargo, existen pocos trabajos experimentales que hayan abordado esta cuestión. En esta tesis, se analiza el efecto de la heterogeneidad del paisaje sobre la incidencia de los virus y la estructura genética de sus poblaciones. Asimismo, se explora como dichos factores ambientales influyen en la importancia relativa que los principales mecanismos de generación de variabilidad genética (mutación, recombinación y migración) tienen en la evolución de los virus. Para ello se ha usado como sistema los begomovirus que infectan poblaciones de chiltepín (Capsicum annuum var. aviculare (Dierbach) D´Arcy & Eshbaugh) en México. Se analizó la incidencia de diferentes virus en poblaciones de chiltepín distribuidas a lo largo de seis provincias biogeográficas, representando el área de distribución de la especie en México, y localizadas en hábitats con diferente grado de intervención humana: poblaciones sin intervención humana (silvestres); poblaciones toleradas (lindes y pastizales), y poblaciones manejadas por el hombre (monocultivos y huertos familiares). Entre los virus analizados, los begomovirus mostraron la mayor incidencia, detectándose en todas las poblaciones y años de muestreo. Las únicas dos especies de begomovirus que se encontraron infectando al chiltepín fueron: el virus del mosaico dorado del chile (Pepper golden mosaic virus, PepGMV) y el virus huasteco del amarilleo de venas del chile (Pepper huasteco yellow vein virus, PHYVV). Por ello, todos los análisis realizados en esta tesis se centran en estas dos especies de virus. La incidencia de PepGMV y PHYVV, tanto en infecciones simples como mixtas, aumento cuanto mayor fue el nivel de intervención humana en las poblaciones de chiltepín, lo que a su vez se asoció con una menor biodiversidad y una mayor densidad de plantas. Además, la incidencia de infecciones mixtas, altamente relacionada con la presencia de síntomas, fue también mayor en las poblaciones cultivadas. La incidencia de estos dos virus también varió en función de la población de chiltepín y de la provincia biogeográfica. Por tanto, estos resultados apoyan una de las hipótesis XVI clásicas de la Patología Vegetal según la cual la simplificación de los ecosistemas naturales debida a la intervención humana conduce a un mayor riesgo de enfermedad de las plantas, e ilustran sobre la importancia de la heterogeneidad del paisaje a diferentes escalas en la determinación de patrones epidemiológicos. La heterogeneidad del paisaje no solo afectó a la epidemiología de PepGMV y PHYVV, sino también a la estructura genética de sus poblaciones. En ambos virus, el nivel de diferenciación genética mayor fue la población, probablemente asociado a la capacidad de migración de su vector Bemisia tabaci; y en segundo lugar la provincia biogeográfica, lo que podría estar relacionado con el papel del ser humano como agente dispersor de PepGMV y PHYVV. La estima de las tasas de sustitución nucleotídica de las poblaciones de PepGMV y PHYVV mostró una rápida dinámica evolutiva. Los árboles filogenéticos de ambos virus presentaron una topología en estrella, lo que sugiere una expansión reciente en las poblaciones de chiltepín. La reconstrucción de los patrones de migración de ambos virus indicó que ésta expansión parece haberse producido desde la zona central de México siguiendo un patrón radial, y en los últimos 30 años. Es importante tener en cuenta que el patrón espacial de la diversidad genética de las poblaciones de PepGMV y PHYVV es similar al descrito previamente para el chiltepín lo que podría dar lugar a la congruencia de las genealogías del huésped y la de los virus. Dicha congruencia se encontró cuando se tuvieron en cuenta únicamente las poblaciones de hábitats silvestres y tolerados, lo que probablemente se debe a una codivergencia en el espacio pero no en el tiempo, dado que la evolución de virus y huésped han ocurrido a escalas temporales muy diferentes. Finalmente, el análisis de la frecuencia de recombinación en PepGMV y PHYVV indicó que esta juega un papel importante en la evolución de ambos virus, dependiendo su importancia del nivel de intervención humana de la población de chiltepín. Este factor afectó también a la intensidad de la selección a la que se ven sometidos los genomas de PepGMV y PHYVV. Los resultados de esta tesis ponen de manifiesto la importancia que la reducción de la biodiversidad asociada al nivel de intervención humana de las poblaciones de plantas y la heterogeneidad del paisaje tiene en la emergencia de nuevas enfermedades virales. Por tanto, es necesario considerar estos factores ambientales a la hora de comprender la epidemiologia y la evolución de los virus de plantas.XVII SUMMARY Plant viruses play a key role as modulators of the spatio-temporal dynamics of their host populations, due to their negative impact in plant fitness. Knowledge on the genetic and environmental factors that determine the epidemiology and the genetic structure of virus populations may help to understand the ecological role of viral infections. However, few experimental works have addressed this issue. This thesis analyses the effect of landscape heterogeneity in the prevalence of viruses and the genetic structure of their populations. Also, how these environmental factors influence the relative importance of the main mechanisms for generating genetic variability (mutation, recombination and migration) during virus evolution is explored. To do so, the begomoviruses infecting chiltepin (Capsicum annuum var. aviculare (Dierbach) D'Arcy & Eshbaugh) populations in Mexico were used. Incidence of different viruses in chiltepin populations of six biogeographical provinces representing the species distribution in Mexico was determined. Populations belonged to different habitats according to the level of human management: populations with no human intervention (Wild); populations naturally dispersed and tolerated in managed habitats (let-standing), and human managed populations (cultivated). Among the analyzed viruses, the begomoviruses showed the highest prevalence, being detected in all populations and sampling years. Only two begomovirus species infected chiltepin: Pepper golden mosaic virus, PepGMV and Pepper huasteco yellow vein virus, PHYVV. Therefore, all the analyses presented in this thesis are focused in these two viruses. The prevalence of PepGMV and PHYVV, in single and mixed infections, increased with higher levels of human management of the host population, which was associated with decreased biodiversity and increased plant density. Furthermore, cultivated populations showed higher prevalence of mixed infections and symptomatic plants. The prevalence of the two viruses also varied depending on the chiltepin population and on the biogeographical province. Therefore, these results support a classical hypothesis of Plant Pathology stating that simplification of natural ecosystems due to human management leads to an increased disease risk, and illustrate on the importance of landscape heterogeneity in determining epidemiological patterns. Landscape heterogeneity not only affected the epidemiology of PepGMV and PHYVV, but also the genetic structure of their populations. Both viruses had the highest level of genetic differentiation at the population scale, probably associated with the XVIII migration patterns of its vector Bemisia tabaci, and a second level at the biogeographical province scale, which could be related to the role of humans as dispersal agents of PepGMV and PHYVV. The estimates of nucleotide substitution rates of the virus populations indicated rapid evolutionary dynamics. Accordingly, phylogenetic trees of both viruses showed a star topology, suggesting a recent diversification in the chiltepin populations. Reconstruction of PepGMV and PHYVV migration patterns indicated that they expanded from central Mexico following a radial pattern during the last 30 years. Importantly, the spatial genetic structures of the virus populations were similar to that described previously for the chiltepin, which may result in the congruence of the host and virus genealogies. Such congruence was found only in wild and let-standing populations. This is probably due to a co-divergence in space but not in time, given the different evolutionary time scales of the host and virus populations. Finally, the frequency of recombination detected in the PepGMV and PHYVV populations indicated that this mechanism plays an important role in the evolution of both viruses at the intra-specific scale. The level of human management had a minor effect on the frequency of recombination, but influenced the strength of negative selective pressures in the viral genomes. The results of this thesis highlight the importance of decreased biodiversity in plant populations associated with the level of human management and of landscape heterogeneity on the emergence of new viral diseases. Therefore it is necessary to consider these environmental factors in order to fully understand the epidemiology and evolution of plant viruses.

Phytostabilization of metals in mine soils using Brassica juncea in combination with organic amendments

Background and aims The high metal bioavailability and the poor conditions of mine soils yield a low plant biomass, limiting the application of phytoremediation techniques. A greenhouse experiment was performed to evaluate the effects of organic amendments on metal stabilization and the potential of Brassica juncea L. for phytostabilization in mine soils. Methods Plants were grown in pots filled with soils collected from two mine sites located in Central Spain mixed with 0, 30 and 60 tha?1 of pine bark compost and horse- and sheep-manure compost. Plant biomass and metal concentrations in roots and shoots were measured. Metal bioavailability was assessed using a rhizosphere-based method (rhizo), which consists of a mixture of low-molecular-weight organic acids to simulate root exudates. Results Manure reduced metal concentrations in shoots (10?50 % reduction of Cu and 40?80 % of Zn in comparison with non-amended soils), bioconcentration factor (10?50 % of Cu and 40?80 % of Zn) and metal bioavailability in soil (40?50 % of Cu and 10?30 % of Zn) due to the high pH and the contribution of organic matter. Manure improved soil fertility and was also able to increase plant biomass (5?20 times in shoots and 3?30 times in roots), which resulted in a greater amount of metals removed from soil and accumulated in roots (increase of 2?7 times of Cu and Zn). Plants grown in pine bark treatments and in non-amended soils showed a limited biomass and high metal concentrations in shoots. Conclusions The addition of manure could be effective for the stabilization of metals and for enhancing the phytostabilization ability of B. juncea in mine soils. In this study, this species resulted to be a potential candidate for phytostabilization in combination with manure, differing from previous results, in which B. juncea had been recognized as a phytoextraction plant.

Improvement of root architecture under abiotic stress through control of auxin homeostasis in Arabidopsis and Brassica crops

Auxin plays an important role in many aspects of plant development including stress responses. Here we briefly summarize how auxin is involved in salt stress, drought (i.e. mostly osmotic stress), waterlogging and nutrient deficiency in Brassica plants. In addition, some mechanisms to control auxin levels and signaling in relation to root formation (under stress) will be reviewed. Molecular studies are mainly described for the model plant Arabidopsis thaliana, but we also like to demonstrate how this knowledge can be transferred to agriculturally important Brassica species, such as Brassica rapa, Brassica napus and Brassica campestris. Moreover, beneficial fungi could play a role in the adaptation response of Brassica roots to abiotic stresses. Therefore, the possible influence of Piriformospora indica will also be covered since the growth promoting response of plants colonized by P. indica is also linked to plant hormones, among them auxin.

Further expansion of the alien seaweed Caulerpa taxifolia var. distichophylla (Sonder) Verlaque, Huisman & Procacini (Ulvophyceae, Bryopsidales) in the Eastern Mediterranean Sea

Date of Acceptance: 27/04/2015 We are grateful to Andreas Antoniou (Dep. of Environment, Ministry of Agriculture, Rural Development & Environment, Cyprus) for his assistance in the preparation of the illustrations. We would also like to thank Dr. Sotiris Orfanidis (NAGREF – Fisheries Research Institute, Kavala, Greece) for his valuable advice and both the DFMR and HSR / HCMR Rhodes crew and George Hatiris for their help in samplings. Special thanks are due to Dinos Leonidou (SeaQuest Divers Cyprus) for accompanying the deep dive for sampling Caulerpa at Cavo Greco. We are grateful to the Total Foundation (Paris) for its funding support to this study within the framework of the project “Brown algal ecology and biodiversity in the eastern Mediterranean Sea” and to the MASTS pooling initiative (Marine Alliance for Science and Technology for Scotland, funded by the Scottish Funding Council and contributing institutions; grant reference HR09011).

Isolation and characterization of neutral-lipid-containing organelles and globuli-filled plastids from Brassica napus tapetum

The monolayer tapetum cells of the maturing flowers of Brassica napus contain abundant subcellular globuli-filled plastids and special lipid particles, both enriched with lipids that are supposed to be discharged and deposited onto the surface of adjacent maturing pollen. We separated the two organelles by flotation density gradient centrifugation and identified them by electron microscopy. The globuli-filled plastids had a morphology similar to those described in other plant species and tissues. They had an equilibrium density of 1.02 g/cm3 and contained neutral esters and unique polypeptides. The lipid particles contained patches of osmiophilic materials situated among densely packed vesicles and did not have an enclosing membrane. They exhibited osmotic properties, presumably exerted by the individual vesicles. They had an equilibrium density of 1.05 g/cm3 and possessed triacylglycerols and unique polypeptides. Several of these polypeptides were identified, by their N-terminal sequences or antibody cross-reactivity, as oleosins, proteins known to be associated with seed storage oil bodies. The morphological and biochemical characteristics of the lipid particles indicate that they are novel organelles in eukaryotes that have not been previously isolated and studied. After lysis of the tapetum cells at a late stage of floral development, only the major plastid neutral ester was recovered, whereas the other abundant lipids and proteins of the two tapetum organelles were present in fragmented forms or absent on the pollen surface.

The pollen determinant of self-incompatibility in Brassica campestris

Many flowering plants possess self-incompatibility (SI) systems that prevent inbreeding. In Brassica, SI is controlled by a single polymorphic locus, the S locus. Two highly polymorphic S locus genes, SLG (S locus glycoprotein) and SRK (S receptor kinase), have been identified, both of which are expressed predominantly in the stigmatic papillar cell. We have shown recently that SRK is the determinant of the S haplotype specificity of the stigma. SRK is thought to serve as a receptor for a pollen ligand, which presumably is encoded by another polymorphic gene at the S locus. We previously have identified an S locus gene, SP11 (S locus protein 11), of the S9 haplotype of Brassica campestris and proposed that it potentially encodes the pollen ligand. SP11 is a novel member of the PCP (pollen coat protein) family of proteins, some members of which have been shown to interact with SLG. In this work, we identified the SP11 gene from three additional S haplotypes and further characterized the gene. We found that (i) SP11 showed an S haplotype-specific sequence polymorphism; (ii) SP11 was located in the immediate flanking region of the SRK gene of the four S haplotypes examined; (iii) SP11 was expressed in the tapetum of the anther, a site consistent with sporophytic control of Brassica SI; and (iv) recombinant SP11 of the S9 haplotype applied to papillar cells of S9 stigmas, but not of S8 stigmas, elicited SI response, resulting in inhibition of hydration of cross-pollen. All these results taken together strongly suggest that SP11 is the pollen S determinant in SI.

The incompatible interaction between Phytophthora parasitica var. nicotianae race 0 and tobacco is suppressed in transgenic plants expressing antisense lipoxygenase sequences

Nicotiana tabacum 46-8 cultivar displays an incompatible interaction with race 0 of Phytophthora parasitica var. nicotianae (Ppn), a fungal pathogen of most tobacco cultivars. At the plant level, incompatibility is characterized by the induction of lipoxygenase (LOX, EC = 1.13.11.12) activity and localized hypersensitive cell death before defense gene activation. To evaluate the involvement of LOX in the onset of plant defense, tobacco 46-8 plants were genetically engineered using full-length or partial-length antisense (AS) tobacco LOX cDNA constructs. AS expression strongly reduced elicitor- and pathogen-induced LOX activity. Eight independent AS-LOX lines were selected and assayed for their response to Ppn. After root or stem inoculation with race 0, all AS-LOX lines but one displayed a compatible phenotype whereas control transformed plants, not containing the AS-LOX cassette, showed the typical incompatible reaction. The presence of the fungus in transgenic lines was demonstrated by PCR amplification of a Ppn-specific genomic sequence. A linear relationship was found between the extent of LOX suppression and the size of the lesion caused by the fungus. The AS-LOX plants also showed enhanced susceptibility toward the compatible fungus Rhizoctonia solani. The results demonstrate the strong involvement of LOX in the establishment of incompatibility in plant–microorganism interactions, consistent with its role in the defense of host plants.

Independent deletions of a pathogen-resistance gene in Brassica and Arabidopsis

Plant disease resistance (R) genes confer race-specific resistance to pathogens and are genetically defined on the basis of intra-specific functional polymorphism. Little is known about the evolutionary mechanisms that generate this polymorphism. Most R loci examined to date contain alternate alleles and/or linked homologs even in disease-susceptible plant genotypes. In contrast, the resistance to Pseudomonas syringae pathovar maculicola (RPM1) bacterial resistance gene is completely absent (rpm1-null) in 5/5 Arabidopsis thaliana accessions that lack RPM1 function. The rpm1-null locus contains a 98-bp segment of unknown origin in place of the RPM1 gene. We undertook comparative mapping of RPM1 and flanking genes in Brassica napus to determine the ancestral state of the RPM1 locus. We cloned two B. napus RPM1 homologs encoding hypothetical proteins with ≈81% amino acid identity to Arabidopsis RPM1. Collinearity of genes flanking RPM1 is conserved between B. napus and Arabidopsis. Surprisingly, we found four additional B. napus loci in which the flanking marker synteny is maintained but RPM1 is absent. These B. napus rpm1-null loci have no detectable nucleotide similarity to the Arabidopsis rpm1-null allele. We conclude that RPM1 evolved before the divergence of the Brassicaceae and has been deleted independently in the Brassica and Arabidopsis lineages. These results suggest that functional polymorphism at R gene loci can arise from gene deletions.

Embryo-Specific Gene Expression in Microspore-Derived Embryos of Brassica napus. An Interaction between Abscisic Acid and Jasmonic Acid1, 2

The induction of napin and oleosin gene expression in Brassica napus microspore-derived embryos (MDEs) was studied to assess the possible interaction between abscisic acid (ABA) and jasmonic acid (JA). Napin and oleosin transcripts were detected sooner following treatment with ABA than JA. Treatment of MDEs with ABA plus JA gave an additive accumulation of both napin and oleosin mRNA, the absolute amount being dependent on the concentration of each hormone. Endogenous ABA levels were reduced by 10-fold after treatment with JA, negating the possibility that the observed additive interaction was due to JA-induced ABA biosynthesis. Also, JA did not significantly increase the uptake of [3H-ABA] from the medium into MDEs. This suggests that the additive interaction was not due to an enhanced carrier-mediated ABA uptake by JA. Finally, when JA was added to MDEs that had been treated with the ABA biosynthesis inhibitor fluridone, napin mRNA did not increase. Based on these results with the MDE system, it is possible that embryos of B. napus use endogenous JA to modulate ABA effects on expression of both napin and oleosin. In addition, JA could play a causal role in the reduction of ABA that occurs during late stages of seed development.