The use of neutral and non-neutral SSRs to analyse the genetic structure of a Tunisian collection of Medicago truncatula lines and to reveal associations with eco-environmental variables.

In this study, we investigated the genetic diversity of a collection of 136 Medicago truncatula lines from 10 Tunisian natural populations collected in well-defined locations and in various ecological conditions of soil, salinity and water availability. The genetic diversity was evaluated using a set of 18 microsatellites (SSRs), representing the 8 chromosomes of M. truncatula. A neutrality test showed that 7 SSRs were non-neutral with evidence of balancing selection. The 11 neutral SSRs revealed a geographical pooling with the Tunisian Dorsale axis restricting migration of alleles. The 7 non-neutral alleles demonstrate a correlation with rainfall, altitude and salinity environmental variables suggesting that these SSRs are linked to genes involved in water use efficiency, resistance to salinity or adaptation to altitude, and that there is local adaptation of M. truncatula to these variables. This demonstrates that the choice of so-called neutral markers should be carefully evaluated in population genetic studies. This study illustrates the genetic diversity occurring in natural Tunisian populations of M. truncatula and describes the first collection of this species dedicated to natural variation involved in adaptation to the environment.

Using a model-based framework for analysing genetic diversity during germination and heterotrophic growth of Medicago truncatula.

BACKGROUND AND AIMS: The framework provided by an emergence model was used: (1) for phenotyping germination and heterotrophic growth of Medicago truncatula in relation to two major environmental factors, temperature and water potential; and (2) to evaluate the extent of genetic differences in emergence-model parameters. METHODS: Eight cultivars and natural accessions of M. trunculata were studied. Germination was recorded from 5 to 30 degrees C and from 0 to -0.75 MPa, and seedling growth from 10 to 20 degrees C. KEY RESULTS: Thermal time to reach 50 % germination was very short (15 degrees Cd) and almost stable between genotypes, while base temperature (2-3 degrees C) and base water potential for germination (-0.7 to -1.3 MPa) varied between genotypes. Only 35 degrees Cd after germination were required to reach 30 mm hypocotyl length with significant differences among genotypes. Base temperature for elongation varied from 5.5 to 7.5 degrees C. Low temperatures induced a general shortening of the seedling, with some genotypes more responsive than others. No relationship with initial seed mass or seed reserve distribution was observed, which might have explained differences between genotypes and the effects of low temperatures. CONCLUSIONS: The study provides a set of reference values for M. trunculata users. The use of the ecophysiological model allows comparison of these values between such non-crop species and other crops. It has enabled phenotypic variability in response to environmental conditions related to the emergence process to be identified. The model will allow simulation of emergence differences between genotypes in a range of environments using these parameter values. Genomic tools available for the model species M. trunculata will make it possible to analyse the genetic and molecular determinants of these differences.

AER1, a major gene conferring resistance to Aphanomyces euteiches in Medicago truncatula.

Aphanomyces euteiches is a major soilborne oomycete pathogen that infects various legume species, including pea and alfalfa. The model legume Medicago truncatula has recently emerged as a valuable genetic system for understanding the genetic basis of resistance to A. euteiches in leguminous crops. The objective of this study was to identify genetic determinants of resistance to a broad host-range pea-infecting strain of A. euteiches in M. truncatula. Two M. truncatula segregating populations of 178 F(5) recombinant inbred lines and 200 F(3) families from the cross F83005.5 (susceptible) x DZA045.5 (resistant) were screened for resistance to A. euteiches. Phenotypic distributions observed suggested a dominant monogenic control of resistance. A major locus associated with resistance to A. euteiches, namely AER1, was mapped by bulk segregant analysis to a terminal end of chromosome 3 in M. truncatula and explained 88% of the phenotypic variation. AER1 was identified in a resistance-gene-rich region, where resistance gene analogs and genes associated with disease resistance phenotypes have been identified. Discovery of AER1 opens up new prospects for improving resistance to A. euteiches in cultivated legumes using a comparative genomics approach.

Keratitis secondary to Fusarium spp. in Spain 2012-2014. / Queratitis secundaria a Fusarium spp. en España 2012-2014.

OBJECTIVE: To describe the clinical-epidemiological characteristics of a case series of fungal keratitis associated with Fusarium spp.., in Spain during the years 2012 to 2014. METHODS: A retrospective study of a case series was conducted on a systematic random sample of 10% of patients identified in Health Centres of provincial capitals (n=250). The centres were asked whether they had been presented with cases of Fusarium spp. keratitis characterised by microbiological methods. Of the 23 centres that responded, 14 had detected cases, with 13 of them accepting to participate, and 11 of them completing the study. The latter being sent a previously validated questionnaire. The variables analysed were: age, gender, habitual residence, profession, disease and medical history (previous systemic and ocular diseases, previous eye surgeries), and their outcomes. RESULTS: The response rate was 92%, identifying 23 cases of Fusarium spp.. of which 21 (91.3%) of them lived in urban areas. The professions most affected by the disease included chefs, administrative, and technical, with 13 cases (56.5%). The treatment guidelines established to confirm the infection showed the combined use of topical antibiotics associated with antiviral and/or antifungal agents. The use of contact lenses (86.9%) was the main risk factor. DISCUSSION: This study showed that this is a rare disease in Spain, but that a large percentage of people who present with the disease are resident in urban areas, and they work in closed environments, focusing attention on microtraumas caused by use of contact lenses.

TE7, An Inefficient Symbiotic Mutant of Medicago truncatula Gaertn. cv Jemalong.

A mutagenesis program using ethylmethane sulfonate on Medicago truncatula Gaertn cv Jemalong, an annual, autogamous and diploid lucerne, permitted the isolation of a mutant (TE7) unable to establish an effective nitrogen-fixing symbiosis, [Nod+Fix-], with Rhizobium meliloti wild-type strains. The mutant phenotype is characterized by an altered infection process that leads to the formation of two kinds of inefficient nodules on the same root system. A certain proportion of the nodules are small, round, and uninfected, with infection threads limited to the outer root cortical cells. Others develop to a normal elongated shape and are infected; bacterial release occurs but the bacteria do not differentiate into bacteroids. The ratio of invaded to uninvaded nodules depends on the bacterial strain used. Throughout the infection process, certain events correlated with the plant defense response against pathogens can be observed: (a) the presence of polyphenolic compounds associated with the walls of infected cells and also with some parts of infection threads in the root cortex; (b) appositions on infection thread walls during the early stage of infection and also within the central tissue of infected nodules; and (c) autophagy of the plant cells that contain released bacteria. Genetic data suggest that the phenotype of TE7 is under monogenic and recessive control; this gene has been designated Mtsym1.

The early nodulin gene MtN6 is a novel marker for events preceding infection of Medicago truncatula roots by Sinorhizobium meliloti.

MtN6 belongs to a series of cDNA clones representing Medicago truncatula genes transcriptionally activated during nodulation by Sinorhizobium meliloti (P. Gamas, F. de Carvalho Niebel, N. Lescure, and J. V. Cullimore, Mol. Plant-Microbe Interact. 9:233-242, 1996). We show here by in situ hybridization that MtN6 transcripts specifically accumulate first at very localized regions in the outer root cell layers, corresponding to outer cortical cells containing preinfection threads. At later stages, MtN6 expression is observed ahead of growing infection threads, including in the infection zone of mature root nodules. Interestingly, regulation of MtN6 is clearly distinct from that of other early nodulins expressed in the same region of the nodule, in terms of response to bacterial symbiotic mutants and to purified Nod factors. We thus suggest that MtN6 represents the first specific marker of a pathway involved in preparation to infection, which is at least partly controlled by Nod factors. Finally, we discuss the intriguing sequence homology shown by MtN6 to a protein from Emericella (Aspergillus) nidulans, FluG, that plays a key role in controlling the organogenesis of conidiophores (B. N. Lee and T. H. Adams, Genes Dev. 8:641-651, 1994).

[Endophthalmos caused by intravitreal caterpillar hairs. Treatment by direct photocoagulation with argon laser]. / Endophtalmie par poils de chenille intravitréens. Traitement par photocoagulation directe au laser à l'argon.

We describe an infrequent case of endophthalmitis due to intraocular caterpillar hairs. We report a notable decrease of inflammation after Argon laser photocoagulation of the caterpillar hairs. We also present optical and electronic microscope photographs of one of the hairs extracted from the patient's conjunctiva.

[Thoracic actinomycosis. Report of 8 cases]. / L'actinomycose thoracique. A propos de 8 observations.

We report 8 cases of thoracic actinomycosis, a disease which is now uncommon owing to the widespread use of antibiotics and which is caused by anaerobic filamentous bacteria living as saprophytes in natural cavities. Recent pathogenetic data, such as propagation by continuity or blood stream, as well as bacteriological and clinical data (mediastino-pulmonary, pleural, parietal, cardiac and disseminated lesions) are reviewed. Diagnostic problems are due to the difficulties encountered in trying to isolate the saprophytic organism, and pathological examination is often required for the diagnosis. Treatment is basically medical and consists of penicillin G or A administered for prolonged periods. Nitroimidazoles are ineffective against these anaerobic bacteria.

Queratitis secundaria a Fusarium spp. en España 2012-2014 / Keratitis secondary to Fusarium spp. in Spain 2012-2014

OBJETIVO: Describir las características clinicoepidemiológicas de una serie de casos de queratitis fúngica asociada con Fusarium spp., en España durante los años 2012 a 2014. MÉTODOS: Estudio retrospectivo de una serie de casos. Se identificaron los centros sanitarios que se encontraban en las capitales provinciales (n = 250), obteniéndose una muestra aleatoria sistemática del 10%. Se les preguntó si habían presentado casos de queratitis por Fusarium spp. caracterizados mediante métodos microbiológicos, 23 centros respondieron, detectando casos en 14 de ellos, aceptando participar 13, completando el estudio 11 instituciones, a los que se les envió el cuestionario previamente validado. Las variables analizadas fueron: edad, sexo, residencia habitual, profesión, antecedentes patológicos y médicos (enfermedades sistémicas y oculares previas, cirugías oculares previas) y su evolución. RESULTADOS: La tasa de respuesta fue del 92%, identificando 23 casos de Fusarium spp.; 21 casos (91,3%) vivían en zonas urbanas. Los profesionales fueron los más afectados por la enfermedad (chef, administrativo, técnico) con 13 casos (56,5%). Las pautas de tratamiento establecidas antes de la confirmación de la infección evidenciaron el uso combinado de antibióticos tópicos asociados a agentes antivirales y/o antifúngicos, siendo el principal factor de riesgo el uso de lentes de contacto (86,9%). DISCUSIÓN: Es una enfermedad poco frecuente en nuestro medio, un gran porcentaje de las personas residían en áreas urbanas y su trabajo se realizaba en entornos cerrados, enfocando la atención en los microtraumas causados por el uso de lentes de contacto

OBJECTIVE: To describe the clinical-epidemiological characteristics of a case series of fungal keratitis associated with Fusarium spp.., in Spain during the years 2012 to 2014. METHODS: A retrospective study of a case series was conducted on a systematic random sample of 10% of patients identified in Health Centres of provincial capitals (n = 250). The centres were asked whether they had been presented with cases of Fusarium spp. keratitis characterised by microbiological methods. Of the 23 centres that responded, 14 had detected cases, with 13 of them accepting to participate, and 11 of them completing the study. The latter being sent a previously validated questionnaire. The variables analysed were: age, gender, habitual residence, profession, disease and medical history (previous systemic and ocular diseases, previous eye surgeries), and their outcomes. RESULTS: The response rate was 92%, identifying 23 cases of Fusarium spp.. of which 21 (91.3%) of them lived in urban areas. The professions most affected by the disease included chefs, administrative, and technical, with 13 cases (56.5%). The treatment guidelines established to confirm the infection showed the combined use of topical antibiotics associated with antiviral and/or antifungal agents. The use of contact lenses (86.9%) was the main risk factor. DISCUSSION: This study showed that this is a rare disease in Spain, but that a large percentage of people who present with the disease are resident in urban areas, and they work in closed environments, focusing attention on microtraumas caused by use of contact lenses

Comparison of rhizobia that nodulate Medicago laciniata and Medicago truncatula present in a single Tunisian arid soil.

The rhizobia present in a single arid region Tunisian soil that nodulate Medicago laciniata and Medicago truncatula were compared. All isolates, 40 from each host, were Sinorhizobium meliloti based on 16S rRNA polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) patterns and subsequent confirmation by sequence analysis of the 16S rRNA genes in four representatives from each host species. There was no apparent relationship between Medicago host species of isolation and the nodulating rhizobial genome as determined by repetitive extragenic palandromic PCR. The isolates of M. laciniata were distinguished from those of M. truncatula present in the same soil by variation in PCR-RFLP of nifDK, indicating that this dissimilarity is originally genetic and not geographic. While forming effective symbioses with their own respective isolates, both M. laciniata and M. truncatula formed ineffective true nodules, nodule-like structures, or no nodules at all in cross-inoculation tests, as confirmed by the histological observations.

Functional mapping in pea, as an aid to the candidate gene selection and for investigating synteny with the model legume Medicago truncatula.

The identification of the molecular polymorphisms giving rise to phenotypic trait variability-both quantitative and qualitative-is a major goal of the present agronomic research. Various approaches such as positional cloning or transposon tagging, as well as the candidate gene strategy have been used to discover the genes underlying this variation in plants. The construction of functional maps, i.e. composed of genes of known function, is an important component of the candidate gene approach. In the present paper we report the development of 63 single nucleotide polymorphism markers and 15 single-stranded conformation polymorphism markers for genes encoding enzymes mainly involved in primary metabolism, and their genetic mapping on a composite map using two pea recombinant inbred line populations. The complete genetic map covers 1,458 cM and comprises 363 loci, including a total of 111 gene-anchored markers: 77 gene-anchored markers described in this study, 7 microsatellites located in gene sequences, 16 flowering time genes, the Tri gene, 5 morphological markers, and 5 other genes. The mean spacing between adjacent markers is 4 cM and 90% of the markers are closer than 10 cM to their neighbours. We also report the genetic mapping of 21 of these genes in Medicago truncatula and add 41 new links between the pea and M. truncatula maps. We discuss the use of this new composite functional map for future candidate gene approaches in pea.

Medicago-Sinorhizobium symbiotic specificity evolution and the geographic expansion of Medicago.

The legume genus Medicago interacts with soil bacteria commonly referred to as rhizobia, in a nitrogen fixing symbiosis. We analysed the diversity of symbiotic association specificity among the two organisms, and its evolution in the plant genus. Nitrogen fixation tests and molecular phylogenetic reconstructions revealed that the genus Medicago includes more symbiotic specificity groups than previously suggested and that plant specificity is highly unstable and has repeatedly switched along the diversification of this genus. A phylogenetic analysis including geographical data shows that bacterial geographical diversity distribution has a strong influence on the geographic distribution of plant species and their ability to colonize new areas. Multiple other modifications of specificity occurred along the diversification of the genus, presumably due to selection for specialization to a single bacterial biovar. Codivergence between plants and bacteria may also have taken place.

Cross-species amplification of Medicago truncatula microsatellites across three major pulse crops.

Model plants are facilitating the genetic characterization and comparative mapping of a number of traditional crops. Medicago truncatula has been widely accepted as a model plant to this end as it provides the essential tools for multiple aspects of legume genetics and genomics. A large set of markers from highly conserved M. truncatula gene regions is being created and used to establish a worldwide framework for comparative genomic studies in legumes. We have investigated the potential for cross-species amplification of 209 expressed sequence tag (EST)-based and 33 bacterial artificial chromosome (BAC)-based microsatellites from M. truncatula in the three most important European legume pulses-pea, faba bean and chickpea-that might facilitate future comparative mapping. Our results revealed significant transferability of M. truncatula microsatellites to the three pulses (40% in faba bean, 36.3% in chickpea and 37.6% in pea). The percentage of M. truncatula EST-SSRs (simple sequence repeats) amplified in the three crops (39-43%) was twofold higher than that of the genomic SSRs (21-24%). Sequence analysis determined that the level of conservation in the microsatellite motif was very low, while the flanking regions were generally well conserved. The variations in the sequences were mainly due to changes in the number of repeat motifs in the microsatellite region combined with indel and base substitutions. None of the functional microsatellites showed direct polymorphism among the parental genotypes tested, consequently preventing their immediate use for mapping purposes.

[Double-stranded sequences in RNA transcribed in vitro by E. coli RNA polymerase on wheat nuclear DNA]. / Séquences en double chaîne dans l'ARN transcrit in vitro par la RNA polymérase de E. coli sur l'ADN nucléaire de blé

RNA obtained through the transcription of wheat DNA by E. coli RNA polymerase was examined. This RNA contains double chain sequences which, after thermal denaturation, show instantaneous reassociation. A "hair-pin" like structure is proposed for this type of sequence.

Plant gene expression during effective and ineffective nodule development of the tropical stem-nodulated legume Sesbania rostrata.

The expression of plant genes during symbiosis of Sesbania rostrata with Rhizobium sp. and Azorhizobium caulinodans was studied by comparing two-dimensional PAGE patterns of in vitro translation products of poly(A)(+) RNA from uninfected roots and stems with that of root and stem nodules. Both types of nodules are essentially similar, particularly when stem nodules are formed in the dark. We detected the specific expression of at least 16 genes in stem and root nodules and observed the stimulated expression of about 10 other genes in both nodules. Six of the nodule-specific translation products (apparent molecular masses around 16 kDa) cross-react with an antiserum raised against leghemoglobin purified from Sesbania rostrata stem nodules. During stem nodule development, most of the nodule-stimulated genes are expressed concomitantly with leghemoglobin at day 12 after inoculation. However, some genes are already stimulated at days 6-7, some others later in development (day 18), and some are transiently activated. Patterns of root nodules induced by either Azorhizobium caulinodans strain ORS571, capable of effective root and stem nodulation, or Rhizobium sp. strain ORS51, capable of effective root nodulation only, are very similar except for a specific 37.5 kDa polypeptide. Several types of ineffective stem and root nodules were studied; in every case the amount of leghemoglobin components appeared reduced together with most of the nodule-stimulated polypeptides.